Therapeutic applications of clenbuterol

ABSTRACT

The present invention relates to therapeutic uses of clenbuterol in humans and animals. The uses include retarding or reversing muscle disease such as muscular dystrophy, alleviating or reversing peripheral nervous system disease, and alleviating or reversing central nervous system disease.

This Application is a Divsional of U.S. application Ser. No. 827,839filed Jan. 29, 1992, now abandoned, which is a continuation-in-part ofapplication Ser. No. 133,702 filed Dec. 16 1987, now abandoned.

This invention relates to therapeutic applications of beta-adrenergicagonists, and more particular but not exclusively to increasing thegrowth of innervated muscles in animals and humans, to retarding orreversing atrophy of denervated muscles in animals and humans, toalleviating or reversing the effects of various diseases in humans andanimals, to modification of foetal and neonatal growth in animals andhumans, and to possible genetic modification of the developing foetus ofan animal or a human.

(References to "foetus" are to be taken as non-exclusively referring tothe unborn offspring of humans and of non-human mammals, and to extendto the embryos of non-mammalian creatures to the extent that thesubstances and procedures herein described are effective.)

There are circumstances in which it is desirable that a live bodycomprising innervated muscles be fed so as to enhance the deposition ofbody protein rather than fat. Further, in a live body which has beeninjured by accident, disease, or surgery so that muscles are denervated,it is desirable that the usual consequential atrophy of the denervatedmuscles be inhibited prior to its occurrence, or reversed if atrophy hasalready occurred.

According to a first aspect of the invention there is provided the useof a beta-adrenergic agonist in biocompatible form, and selected toenhance one or more muscular properties comprising weight, fibre number,and protein content.

According to a second aspect of the invention there is provided abeta-adrenergic agonist in biocompatible form, and selected to reducefat deposition from dietary input.

According to a third aspect of the invention there is provided abeta-adrenergic agonist in biocompatible form, and selected to enhancemuscle growth while reducing fat deposition from dietary input.

In the first, second and third aspects of the invention, thebeta-adrenergic agonist is preferably a beta sympathomimetic agent inall or part of its functions.

In the first, second, and third aspects of the invention the preferredbeta-adrenergic agonist is clenbuterol, which is (4-amino-3,5-dichlorophenyl)-2-tertbutyl-amino ethanol hydrochloride, ie abiocompatible acid addition salt of the compound whose formula isillustrated in the claim of Federal German Offenlegungsschrift 1793416.

For the purposes of one or more of the first, second and third aspectsof the invention, alternative beta-adrenergic agonists are:

Isoetharine, which is 1-(3,4-dihydroxyphenol)-2-isopropyl amino ethanolhydrochloride;

Orciprenaline, which is ±-1-(3,5-dihydroxyphenyl)-2-isopropyl aminoethanol sulphate;

Reproterol, which is 7-(-((3,5, betatrihydroxyphenyl)amino)propyl)theophylline;

Salbutamol, which is2-tertbutylamino-1-(4-hydroxy-3-hydroxymethylphenyl) ethanol sulphate;

Terbutaline, which is 2-tert-butylamino-1-(3,5-dihydroxyphenyl) ethanolsulphate;

Fenoterol, which is 1,3, benzenediol,5-[1-hydroxy-2-[[2-(4-hydroxyphenyl)-1-methylethyl]amino]ethyl;

Cimaterol, which is benzonitrile,2-amino-5-[1-hydroxy-2-[(1-methylethyl)amino]ethyl]; and

Ractopamine, which is[1-(4-hydroxyphenyl)-2(1-methyl-3-(4-hydroxyphenyl))propylamino]ethanol.

The invention also comprises effective analogues of the above-listedbeta-adrenergic agonists.

For anabolic effects on skeletal muscle, clenbuterol is the mostfavoured beta-adrenergic agonist, while orciprenaline, isoetharine,fenoterol, cimaterol, and ractopamine are considered also to havesignificant anabolic effects in one or more forms of muscle.

For catabolic effects on body fat, clenbuterol is the most favouredbeta-adrenergic agonist, while others of the listed beta-adrenergicagonists are considered also to have potentially significant cataboliceffects on body fat.

Of the above-listed beta-adrenergic agonists that were specificallystudied, only clenbuterol markedly increased the mass of leg muscles intests on rats, while orciprenaline and isoetharine both produced somesimilar effect of lesser extent. Isoetharine and salbutamolsignificantly increased cardiac mass of rats, while terbutaline andreproterol reduced hepatic mass. With the exception of orciprenaline,all the beta-adrenergic agonists increased the mass of interscapularbrown fat pads in rats. Orciprenaline and isoetharine increased bodyprotein mass in rats, although not as effectively as clenbuterol.Terbutaline and orciprenaline also reduced body fat mass in rats, butnot as effectively as clenbuterol. A significant relationship wasdiscovered between decreases in body fat and hepatic mass, while changesin leg muscle mass correlated significantly with the increase in bodyprotein.

The inventors have made a further surprising discovery in that theundesirable side-effects of the beta-adrenergic agonists that haveanabolic properties can be substantially counteracted by thesimultaneous use of a suitable proportion of a beta-adrenergicantagonist, preferably a mixed beta-adrenergic antagonist, but withoutterminating all the therapeutic properties of the beta-adrenergicagonist (some of which might be diminished without necessarily ceasing).

The beta-adrenergic antagonist may be propranolol, atenolol, or HoffmanLa Roche compound Ro 22-4574. The preferred combination is clenbuterol(or a clenbuterol analogue) and propranolol.

According to a fourth aspect of the invention there is provided a noveluse of a beta-adrenergic agonist for alleviating or reversing loss offunction of striated muscle arising acutely or chronically or otherwisefrom one or more of the following illnesses:

a. muscle disease;

b. peripheral nervous system disease;

c. central nervous system disease;

d. a humorally mediated catabolic state;

e. trauma arising from accident;

f. trauma arising from surgery;

g. atrophy arising from temporary disuse.

By way of examples of the above medical indications, the followingspecific indications are mentioned:

1. in respect of muscle disease; retardation of the progressivedeterioration of muscular function in muscular dystrophies;

2. alleviation or reversal of neurological disease in the form of lowermotor neurone--motor neurone disease, traumatic or inflammatorymononeuritides (such as radial nerve palsy, Bell's palsy, and similarillnesses), and polyneuropathy (such as Guillain-Barre Syndrome);

3. in respect of neurological disease in the form of upper motor neuronedisease, limitation of wasting following cerebrovascular accidents (suchas strokes) and to improve muscle function in partially paralysed limbs,and improvement of muscle bulk and muscle quality in paraplegia orquadriplegia, especially where external electrical stimulation is to beused (for example to induce walking by use of a transcutaneous orpercutaneous electromyoprosthesis);

4. alleviation or retardation of cachectic conditions --in malignantdiseases (including cancer cachexia) to delay deterioration inwell-being associated with loss of muscle bulk, in cases of acquiredimmune deficiency syndrome, and to limit muscle breakdown in states ofsepsis or after major trauma;

5. utilisation of muscle-specific protein anabolic effects andconcomitant reduced protein gain in the gastrointestinal tract to effecta reduction of nutrient supply (particularly protein) to intestinaltumours, and thereby limit their growth;

6. in perioperative circumstances, to reduce the negative nitrogenbalance and muscle protein loss associated with surgery both in ageneral (whole-body) sense and to limit muscle loss around operativesites, for example (i) to preserve functional muscle in elderly subjectshaving surgery for fractured neck of femur; (ii) to reduce risk ofincisional hernias after abdominal surgery; (iii) to improve healing andrehabilitation of amputees by better preservation of functional tissuein muscle flaps;

7. limitation of atrophy arising from temporary disuse as inneurological disease (see 2 and 3 above) and in states of intentionalimmobilisation, for example limbs in plaster casts and thigh musclesafter cartilage operations;

8. for medical benefit in the case of combined medical indications ofadvanced respiratory disease and poor respiratory musculature presentingtogether and where increased bulk of respiratory muscles may aidrespiratory function, with potential double benefit if thebeta-adrenergic agonist selected for beneficial effects on musculatureis also a bronchodilator (as is notably the case where thebeta-adrenergic agonist is clenbuterol which is indicated for treatmentof bronchospasm);

9. for growth promotion in presentations of wasting illnesses such ascystic fibrosis and cerebral palsy in children (where the children tendto grow poorly and to develop little muscle).

In respect of all the medical indications comprised within the fourthaspect of the invention, the preferred beta-adrenergic agonist isclenbuterol (or an effective analogue of clenbuterol), but otherbeta-adrenergic agonists may alternatively or additionally be employed,such as those mentioned in connection with the first, second, and thirdaspects of the present invention.

According to a fifth aspect of the present invention there is provided anovel use of a beta-adrenergic agonist for use as a medicament formodifying the growth of foetal and/or neonatal animals and humans.

In the fifth aspect of the invention, the preferred beta-adrenergicagonist is clenbuterol (or an effective analogue of clenbuterol), butother beta-adrenergic agonists may be used as an alternative to (or inaddition to) clenbuterol and/or clenbuterol analogues, such as thosementioned in connection with the first, second, and third aspects of theinvention. It has been discovered that the administration of clenbuterolto at least one species of mammal when pregnant or lactating producesthe following surprising results:

i. muscle growth is caused in the dam (female parent) despite thephysiological drain of pregnancy or lactation;

ii. a reduction in muscle fibre number but a converse increase in musclefibre size is caused in the foetal or neonatal offspring of thepregnant/lactating dam; and

iii. these effects in the offspring are apparently persistent.

According to a sixth aspect of the invention there is provided a noveluse of a beta-adrenergic agonist as a medicament for geneticmodification of the developing foetus of an animal or human. The geneticmodification which is particularly but non-exclusively intended is toaffect the developing foetus at its specific stage of development atwhich the dystrophic gene is expressed, so as to obviate or mitigate asubsequent occurrence of muscular dystrophy. The preferredbeta-adrenergic agonist in this sixth aspect of the invention isclenbuterol (or an effective analogue of clenbuterol), but otherbeta-adrenergic agonists may be used as an alternative to (or inaddition to) clenbuterol and/or clenbuterol analogues, such as thosementioned in connection with the first, second and third aspects of theinvention.

According to a seventh aspect of the invention, there is provided amethod of increasing the muscular performance of a human or animal, saidmethod comprising the step of administering to the human or animal aneffective amount of a biocompatible beta-adrenergic agonist selected inaccordance with the first, second or third aspects of the invention. Thehuman or animal may be a participant in a sporting event, and such aparticipant may be an athlete, or a sporting animal, for example aracehorse or a greyhound. The human or animal may additionally oralternatively be desired to have improved muscular properties in anon-sporting context as, for example, in para-military situations andfor draught animals. Thus the seventh aspect of the present inventiondiffers from the other aspects of the invention in that the recipientsof the "treatment" may be healthy and free of significant medicalindications.

Without intent that the following postulations should have any limitingeffect on the invention, it is considered that the novel response ofdenervated muscle to the selected beta-adrenergic agonist involves atleast two mechanisms. The first mechanism involves, in thedown-regulated denervated state, changes in translational efficiency andcapacity which are reminiscent of the classical pleiotypic responses ofcells to growth factors. The second mechanism is a specific reduction inprotein breakdown. With muscles which are in the innervated state, thefirst mechanism is not observed because the muscles have alreadyresponded to other stimuli, whereas the second mechanism is manifested.It might be considered that the difference in response of innervated anddenervated muscle to clenbuterol was associated with increased numbersof beta-adrenergic receptors in the denervated muscle membrane, but thisdoes not appear to be the case, since even in the presence of the mixedbeta-antagonist propranolol in a dose which has been shown to block thecardiac hypertrophy and the reduction in body fat, clenbuterol remainseffective in reversing the muscle protein loss following denervation.This could suggest clenbuterol may be working through a differentreceptor to the beta-one and beta-two receptors susceptible topropranalol.

Embodiments of the invention will now be described by way of example.The following exemplary description is divided into eight sections, eachdealing with a different set of effects on muscles or on muscle relatedmedical indications of one or more beta-adrenergic agonists (eitheralone or in combination with a beta-adrenergic antagonist). Each ofthese eight sections is further sub-divided into several headedsub-sections with self-explanatory sub-headings.

Section 1 The Effect of a Growth Promoting Drug, Clenbuterol, in FibreFrequency and Area in Hind Limb Muscles from Young Male Rats Summary

The effect of dietary administration of clenbuterol in soleus andextensor digitorum longus muscles was studied after 4 and 21 days. Bothmuscles showed an increase in wet weight with no significant change intotal fibre number. After 4 days fibre cross-sectional areas wereincreased in soleus, but not in extensor digitorum longus, and after 21days there was a change in fibre frequencies in extensor digitorumlongus but not soleus muscles.

Introduction

Recently there has been considerable interest in ways of manipulatinggrowth and enhancing the deposition of body protein rather than fat.This has led to an examination of the effect on skeletal muscle of avariety of agents which promote growth including drugs such as the betaadrenergic agonist clenbuterol.

Previous studies using clenbuterol have characteristically shown anincrease in muscle growth, which has been attributed to modifications ofprotein turnover. Although an overall increase in muscle protein iselicited by the drug, the way in which this is expressed in terms ofchanges in muscle fibre number or size has not been examined.Furthermore, in other situations in which protein turnover is modifiedsuch as by insulin, it has been found that different muscles havedifferent sensitivities to the treatment. Consequently it was ofinterest to establish how the growth response was expressed in skeletalmuscle and whether muscles such as soleus and extensor digitorum longuswith different proportions of fibre types had different sensitivities toclenbuterol.

Materials and Methods

Animals and Feeding Regime

Male Hooded-Lister rats of the Rowett strain were weaned at 19 days ofage and divided into groups of 6 of equal mean weight. Initially theanimals were housed in their group and offered stock rat diet (CRM nuts,Labsure, K&K Greff, Croydon, UK) ad libitum. The rats were weigheddaily, and after 4 days were, if necessary, regrouped so that the groupshad equal mean weights. The rats were then housed individually and fed asemi-synthetic diet (PW3: Pullar and Webster, British Journal ofNutrition, Volume 37, pages 355-363, 1977) ad libitum for 3 days. Thegroups of animals were then either maintained on PW3 containing noclenbuterol (control groups), or fed PW3 containing the drug at 2 mg/kgdiet (clenbuterol groups). Food intake and body weights were measureddaily for the study which lasted either 4 or 21 days.

Histochemistry

The animals were killed by cervical dislocation and the soleus andextensor digitorum longus muscles from the left hind limb were rapidlyremoved. The mid-belly portion of each muscle was isolated and orientedfrom transverse sectioning. The sample from each muscle was placed on asmall piece of cork, surrounded with optimal cutting temperaturecompound, covered in talcum powder and frozen in liquid nitrogen. Serialtransverse cryostat sections were cut and stained for one of thefollowing: Ca(2+)-activated myofibrillar ATPase, nicotinamide adeninedinucleotide diaphorase, L-glucan phosphorylase oralpha-glycerophosphate dehydrogenase menadione linked. The fibre typeprofile of each muscle was established on the basis of the musclestaining characteristics with each of the above stains (see Table 1).Both soleus and extensor digitorum longus muscles from control ratscomprised three fibre types: fast twitch glycolytic (FG); fast twitchoxidative glycolytic (FOG); and slow twitch oxidative (SO), whichoccurred in different proportions in each muscle type. The percentagefrequency of each fibre type was estimated from a sample of no less than200 fibres per muscle. The cross sectional area of each fibre type wasmeasured using a Hipad digitizing tablet (Bausch and Lomb, Austin, Tex.)linked to a Prime 550 computer (Prime Computer Inc., Framingham,Massachusetts) programmed to calculate the area delineated on thetablet. The mean percentage area occupied by each fibre type wasestimated by multiplying the mean areas of each fibre type by theirpercentage frequency to obtain a total, and expressing the individualproducts as a percentage of that total. The total number of fibrescomprising the whole muscles was determined from photographic prints ofwhole muscle cross sections stained for myofibrillar ATPase.

Sudan black was used to stain for intramuscular lipid in muscles fromboth groups after 4 to 21 days on the diet.

                  TABLE 1                                                         ______________________________________                                        Histochemical description of fibre types in                                   rat muscle                                                                                       Fast                                                                Slow      twitch    Fast     Fast                                             twitch    oxidative twitch   twitch                                  Enzyme   oxidative glycolytic                                                                              glycolytic                                                                             oxidative                               stained  (SO)      (FOG)     (FG)     (FO)                                    ______________________________________                                        Ca (2+)  +         +++       ++       ++ (+)                                  myofib-                                                                       rillar                                                                        ATPase                                                                        NADH-    ++        ++ (+)    +        ++                                      Diaphor-                                                                      ase                                                                           alpha-   +         + (+)     +++      +                                       Glycero-                                                                      phosphate                                                                     dehydro-                                                                      genase                                                                        L-glucan +         ++        +++      +                                       phosph-                                                                       orylase                                                                       ______________________________________                                    

Reagents

Reagents for histochemistry were obtained from Sigma (Poole, Dorset,UK), or BDH (Poole, Dorset, UK). Clenbuterol was obtained fromBoehringer-Ingelheim (Bracknell, Berks., UK).

Statistics

All data are expressed as means with standard deviations. Statisticalanalysis was achieved by the use of a two-tailed t-Test in which, whereappropriate, unequal variances could be taken into account.

RESULTS

The muscles from clenbuterol-treated animals had a greater wet weightthan those from controls (Table 2). With the exception of extensordigitorum longus muscles at 4 days (P=0.06), this difference wassignificant at both times, but the effect was most pronounced in soleusat 4 days. A study of histochemical fibre types and fibre areas insoleus and extensor digitorum longus muscles from clenbuterol-treatedand control animals after 4 or 21 days on the experiment is shown inTable 2.

Soleus

After only 4 days of drug treatment there was a considerable increase inthe mean cross sectional area of the fibres in muscles fromclenbuterol-treated rats. This increase in size over the controls wasonly significant in FOG and SO fibre types: while FG fibres did appearlarger than in controls, the difference was not significant. There wasno difference between muscles from control and clenbuterol-treatedanimals with respect to either the percentage frequency or thepercentage area of any of the fibre types. After 21 days on the dietthere was still a difference in fibre size between control and treatedgroups, but it was no longer significant. Thus the observed response toclenbuterol was maximal during the first 4 days of treatment, and thesubsequent growth rates between 4 and 21 days were similar in the twogroups. However, the calculation of mean percentage area (reflectingfunctional area of one particular fibre type) at 21 days revealed asignificant increase for FOG and a significant decrease for SO fibresrelative to controls.

Extensor Digitorum Longus

After 4 days on the diet there was no statistically significantdifference between the control and treated groups with respect to any ofthe parameters examined. The data for SO fibres do not meritsignificance owing to the small sample size: out of the 6 treatedmuscles examined, SO fibres were demonstrated in only two. After 21 dayson the diet, the small increases in fibre areas with clenbuteroltreatment were not significant, but there were significant changes infibre type proportions. These were expressed as a statisticallysignificant decrease in FOG frequency and an increase in FG frequency.Both changes were coupled to similar but greater changes in meanpercentage areas. No SO fibres were recorded in extensor digitorumlongus of treated rats, although a few were seen in controls at 21 days.

Fat Content

Following clenbuterol treatment for 4 or 21 days both muscles showedqualitatively less intramuscular fat than the equivalent controls.Investigation of muscle sections demonstrated this point with the use ofSudan black staining to reveal stored lipid. In control animals thesoleus muscles gave the most positive staining in accordance with theirhighly oxidative metabolism. Soleus muscles from clenbuterol-treatedanimals gave very weak staining patterns, indicating a marked reductionin stored lipid. Although a similar relationship could be shown betweenextensor digitorum longus muscles from control and treated groups, thedifference was not so marked.

                                      TABLE 2                                     __________________________________________________________________________    (Section 1).                                                                  The effect of clenbuterol treatment on total fibre number, area and           frequency in muscles of the rat                                               Wet      Total                                                                              Mean area Mean %    Mean %                                      weight   fibre                                                                              (sq μm)                                                                              frequency area                                        Muscle                                                                             (mg)                                                                              number                                                                             FOG FG SO FOG FG SO FOG FG SO                                   __________________________________________________________________________    Soleus                                                                        Control                                                                            64  3011 681 597                                                                              964                                                                              45.5                                                                              2.9                                                                              51.6                                                                             37.6                                                                              2.1                                                                              60.3                                 4 days                                                                             8.3 387  105 82 120                                                                              4.0 1.5                                                                              4.6                                                                              5.2 1.2                                                                              6.0                                  Clen..sup.c                                                                        86.sup.b                                                                          3055 979.sup.c                                                                         747                                                                              1201.sup.a                                                                       43.2                                                                              4.5                                                                              52.2                                                                             39.2                                                                              3.1                                                                              57.7                                 4 days                                                                             12.5                                                                              585  112 154                                                                              193                                                                              5.1 1.1                                                                              4.3                                                                              5.7 1.0                                                                              5.3                                  Control                                                                            195 2709 1658                                                                              1461                                                                             2269                                                                             38.1                                                                              4.0                                                                              58.0                                                                             32.1                                                                              2.9                                                                              65.0                                 21 days                                                                            6.0 837  371 251                                                                              539                                                                              6.4 2.9                                                                              4.1                                                                              9.9 2.2                                                                              8.5                                  Clen.                                                                              230.sup.c                                                                         2470 2196                                                                              2099                                                                             2229                                                                             45.2                                                                              2.3                                                                              52.5                                                                             44.6.sup.a                                                                        2.5                                                                              53.3.sup.a                           21 days                                                                            7.0 653  572 1110                                                                             471                                                                              7.3 2.4                                                                              6.7                                                                              7.9 2.1                                                                              7.9                                  Extensor digitorum longus                                                     Control                                                                            55  3181 556 877                                                                              540                                                                              53.5                                                                              45.9                                                                             1.2                                                                              42.3                                                                              57.0                                                                             1.0                                  4 days                                                                             8.0 722  130 166                                                                              34 3.9 4.6                                                                              0.8                                                                              3.5 4.0                                                                              0.8                                  Clen.                                                                              63  3218 580 978                                                                              901.sup.d                                                                        58.6                                                                              41.4                                                                             0.1.sup.d                                                                        45.8                                                                              54.1                                                                             0.2.sup.d                            4 days                                                                             5.0 514  74  84 138                                                                              5.3 5.2                                                                              0.01                                                                             5.0 5.0                                                                              0.1                                  Control                                                                            157 3044 1046                                                                              1723                                                                             864                                                                              56.9                                                                              40.6                                                                             2.4                                                                              45.5                                                                              52.9                                                                             1.6                                  21 days                                                                            6.0 477  124 239                                                                              75 6.2 7.0                                                                              2.0                                                                              7.0 7.5                                                                              1.4                                  Clen.                                                                              179.sup.c                                                                         3375 1209                                                                              2005                                                                             nr 45.6.sup.b                                                                        65.3.sup.c                                                                       nr 33.8.sup.b                                                                        66.2.sup.c                                                                       nr                                   21 days                                                                            8.0 905  172 359   5.2 5.2   4.3 4.4                                     __________________________________________________________________________     nr = none recorded; .sup.a p < 0.05; .sup.b p < 0.01; .sup.c p< 0.005         All parameters are stated as means with standard deviations, n = 6 for al     groups except .sup.d n= 2 and .sup.c n = 5                               

DISCUSSION

The present study showed that clenbuterol increases the size and proteincontent of rat skeletal muscle, and revealed that the growth-promotingeffects on muscle fibre area were both selective and also decreased withtime. Growth promotion was evident in soleus after 4 days, but haddecreased to insignificance by 21 days. It was less pronounced inextensor digitorum longus at both times.

Differences between muscles were also noted with respect tointramuscular fat. Since clenbuterol acts as a beta adrenergic agonist,the observed decrease in intramuscular fat was not unexpected. Thelipolytic activity of beta agonists is documented, and other studiesusing clenbuterol have reported an overall decrease in body fat.However, it was striking that clenbuterol reduced the fat content ofsoleum much more than extensor digitorum longus. This also suggests aparticular sensitivity of soleus to the action of clenbuterol, and isconsistent with a positive correlation between oxidative capacity andadrenergic receptor density.

The present results suggest that the initial increase in soleus weightat 4 days was due to an increase in the size of the two predominantfibre types, FOG and SO (Table 2). However, these pronounced effects onFOG and SO fibre size seemed to be curiously specific to soleus.Although extensor digitorum longus has a considerable complement of FOGfibres and showed an increase in wet weight after 4 days, there was nosignificant change in fibre area of FOG or any other fibre type.Furthermore, there was no significant change in estimated total fibrenumber in either of the two muscle types. Thus it is probable that anincrease in fibre size, rather than number, was responsible for theincrease in weight of soleus. The explanation for the significantincrease in wet weight of extensor digitorum longus muscles at 21 daysis unclear, but the significantly increased percentage area of FOGfibres (see below), together with the trend towards more fibres, mightimply an increase in total cross-sectional area of these muscles.

Observed increases in muscle wet weight have been associated withincreases in muscle protein deposition. Protein deposition representsthe net balance between the rates of simultaneous protein synthesis anddegradation. Thus the rapid growth response in soleus can be attributedto an increase in synthesis, a decrease in degradation, or a combinationof the two. In mature rats treated with clenbuterol subcutaneously, therates of protein synthesis in gastrocnemius muscles were increased by34% over the controls. However, in a systematic study on which thepresent protocol has been based (the results of which are thereforecomparable to those from the present study), it has been shown thatafter 4 or 21 days of dietary administration of clenbuterol, there wasno difference in synthesis rates in the soleus muscles of young ratscompared with controls. Hence they proposed that the drug was acting todecrease the rate of degradation rather than via an effect on synthesis.This concept is interesting in the context of the present results, sinceit would imply that the rapid increase of fibre area in treated soleusmuscles occurred through a change in protein degradation rate which waseither absent or insignificant in similar fibre types from extensordigitorum longus muscles.

While extensor digitorum longus muscles did not respond significantly totreatment at 4 days, at 21 days there were significant changes inpercentage frequency and percentage area of FOG and FG fibre types(Table 2). Soleus also showed significant, but apparently opposite,changes in these parameters after 21 days (Table 2). However, whenconsidered in physiological terms the data suggest that in both musclesclenbuterol treatment led to an increase in the functional area capableof glycolytic metabolism, accompanied by a decrease in slow twitchfibres. Thus, while the growth-promoting effects of clenbuterol at firstseem specific to soleus, the drug appeared to elicit changes in fibrefrequency which were physiologically comparable between the two muscles.The mechanism and significance of this is unclear, but it might bespeculated that clenbuterol (either directly or indirectly) not onlyaffects growth rate by may also influence the contractile and metabolicproperties of the fibres in both muscles.

Section 2 Inhibition and Reversal of Denervation-Induced Atrophy by theBeta-Agonist Growth promoter, Clenbuterol Summary

Dietary administration of the growth promoter, clenbuterol, ameliorateddenervation-induced atrophy in rat soleus muscles. In acutely denervatedmuscles the drug inhibited the appearance of atrophy, and in chronicallydenervated muscles the atrophy was almost fully reversed. Responses inslow twitch oxidative fibres were particularly marked.

INTRODUCTION

Recent attention on growth promoting agents has focused on a limitednumber of beta2-adrenergic agonists. Previous experiments in which oneof these drugs, clenbuterol, was administered orally to male weanlingrats demonstrated an increase in protein deposition and a reduction inbody fat with little effect on either food intake or body weight. Thegrowth promotion appeared to be muscle-specific and the response wasaccompanied by an increase in fibre size. Detailed histochemicalanalysis showed that muscles such as soleus, in which type 1 fibrespredominate, showing a more rapid and generally more marked response.

Measurements of protein turnover in young rats that had been exposedchronically to clenbuterol suggest that the action of the drug appearedto be mediated by a reduction in muscle protein degradation rather thanan increase in protein synthesis. Consequently, it was of interest toestablish whether a muscle which was undergoing atrophy, in whichproteolysis may be increased, would still respond to the growthpromoting influence of the drug. Denervation-induced atrophy was chosenfor study as this form of atrophy appears to be due largely to anelevation in protein degradation.

MATERIALS AND METHODS

Animals and Experimental Protocol

Male Rowett Hooded Lister Rats of the Rowett strain were weaned at 19 dpost partum and divided into groups of 6 animals of equal mean weight.The initial feeding regime and the dose of clenbuterol was exactly asdescribed previously. The animals were housed in cages with a floor ofwire mesh and their body weights were measured daily. Two experimentswere performed which were based on the induction of denervation atrophyin soleus muscle. The term "atrophy" is used to describe both areduction of muscle protein content and a reduction of fibre size incomparison to the innervated control muscles.

(i) "Acute" denervation. The interaction of simultaneous denervation andadministration of clenbuterol was studied to see if the drug couldprevent the initiation of denervation atrophy. Rats (60±2 g body weight)that had been accustomed to the control diet (PW3) for 4 days, wereanaesthetized with ether and a short length (1 cm) of the left sciaticnerve was removed under aseptic conditions. Over the next 4 days therats were offered the control diet ad libitum or the same diet to whichclenbuterol (2 mg/kg) was added. The animals were then killed.

(ii) "Chronic" denervation. The second experiment was designed to testwhether clenbuterol could restore growth in a muscle that was alreadyexhibiting atrophy. Unilateral sciatic denervation was performed onanimals of the same body weight as in experiment 1 and after the surgerythey were offered the control diet for 3 days. At this time the animalseither continued on the control diet or were offered the clenbuteroldiet for a further 4 days when they were killed.

The animals were killed by cervical dislocation and the soleus musclesfrom both limbs were rapidly dissected, weighed and small samples fromthe mid-belly of the muscle were removed for histochemical analysis. Theremaining muscle was frozen in liquid nitrogen and stored at -20° C.until analysed.

Histochemistry

The histochemical analysis of the muscle sections followed the sameprocedure as described previously. However, in these experiments fibretyping was solely based on the use of the Ca(2+)-activated myofibrillarATPase. The soleus muscles comprised three fibre types: slow twitchoxidative (SO), fast twitch oxidative glycolytic (FOG) and fast twitchglycolytic (FG), which occurred with different frequencies. Since FGfibres occurred with a very low frequency (<5%), in the chronicdenervation experiment they were omitted from estimations of areafrequency and percentage area.

Determination of Muscle Protein

Weighed portions of the muscles were homogenised at 4° C. in 3 ml 0.5Mperchloric acid (PCA) in a motor driven all-glass tissue grinder. Thehomogenate was centrifuged at 3000×g for 15 min and the precipitate waswashed with a further 2 ml PCA. After centrifugation the precipitate wasincubated at 37° C. in 0.3M NaOH for 1 h and portions were assayed forprotein against bovine serum albumin standards.

Statistics

The significance of the differences between mean values was assessed byStudent's two-tailed t-test.

RESULTS

Experiment 1 (Table 1)

After unilateral sciatic nerve section all the animals showed a slightreduction in food intake and weight gain. This growth check was mostmarked in the clenbuterol-treated group and was probably due to thecombination of recovery from surgery and the transient reduction in thefood intake of clenbuterol-treated rats that we have noted previously.This initial growth check may explain the somewhat reduced response toclenbuterol that was observed in the innervated muscles. Neverthelessexposure to clenbuterol produced a significant increase in both theweight and protein content of the innervated soleus.

The denervated muscles were significantly lighter and containedsignificantly less protein than the contralateral innervated muscles ofcontrol and clenbuterol-treated rats. However, treatment withclenbuterol significantly inhibited the full expression of thedenervation-related response. Thus both the weight and protein contentof denervated muscles from clenbuterol-treated rats were significantlygreater than those of denervated muscles from the control animals(p<0.005).

The amelioration of the denervation-related response was particularlystriking in the SO fibres. The cross-sectional area of SO fibres fromdenervated muscles of clenbuterol-treated animals was not significantlydifferent from that of the SO fibres from innervated control muscles.However, while FOG fibres of denervated muscles from clenbuterol-treatedanimals were significantly larger (p<0.005) than the correspondingfibres in denervated muscles from untreated animals, they were stillsignificantly smaller (p<0.01) than the corresponding fibres ininnervated muscles from control animals. The differences in averagefibre cross-sectional area were not accompanied by changes in thefrequency of fibres of different types, but the differences in fibreareas were reflected in significant changes in the functional areaaccounted for by SO and FOG fibres.

Experiment 2 (Table 2)

The innervated muscles showed the expected clenbuterol-induced growthresponse, and denervation led to a significantly lower weight andprotein content. It appeared that the effect of denervation wasprogressive as the denervated soleus muscles from the animals in thisexperiment (ie after 7 d of denervation) were lighter and containedprotein than the soleus muscles after 4 d of denervation in experiment1.

The administration of clenbuterol to these chronically denervatedmuscles causes a significant reversal of the atrophy. This was evidentfrom measurements of both muscle protein content and fibrecross-sectional areas. Thus the denervated muscles from theclenbuterol-treated animals showed a statistically significant (p<0.005)increase in protein content over the untreated denervated muscles. Theincrease in protein was reflected by an increase in fibre area. As inexperiment 1, however, SO fibres appeared to have a greater sensitivityto clenbuterol than FOG fibres. Thus, the SO fibres from denervatedsolei of clenbuterol-treated animals had a mean cross-sectional areawhich was significantly greater (p<0.005) than that of SO fibres fromcontrol denervated muscles. Indeed, the increase in SO fibre area in thedenervated muscles from clenbuterol-treated animals was such that thefibre area had been nearly restored to control innervated values. TheFOG fibres from denervated muscles from clenbuterol-treated animals were

                                      TABLE 1                                     __________________________________________________________________________    (Section 2)                                                                   Experiment 1-The effect on                                                    muscle of simultaneous denervation and administration of clenbuterol          Mean values together with 1 SD indicated in brackets                          Control              Clenbuterol                                              Body Weight (g) 78.7 (1.4)                                                                         Body weight (g) 78.6 (1.7)                               __________________________________________________________________________    Innervated                                                                              Denervated Innervated Denervated                                    Control   Control    Clenbuterol                                                                              Clenbuterol                                   Muscle wet weights (mg)                                                                 ***        ***                                                      31 (3)    24 (1)     40 (2)     31 (2)                                        Muscle protein (mg/g body weight)                                                       ***                   ***                                           0.080 (0.011)                                                                           0.050 (0.006)                                                                            0.086 (0.005)                                                                            0.064 (0.011)                                 Mean cross-sectional areas (sq μm)                                         FOG FG SO FOG FG SO  FOG FF  SO FOG FG  SO                                    874    1085                                                                             368.sup.a                                                                         400                                                                              532.sup.a                                                                         970 900 1109                                                                             668.sup.a                                                                         788 1069.sup.c                            (134)  (40)                                                                             (68)                                                                              (95)                                                                             (113)                                                                             (61)                                                                              (142)                                                                             (63)                                                                             (80)                                                                              (247)                                                                             (132)                                 Mean percentage frequencies                                                   50     50 47.3                                                                              2  51.3                                                                              48.2                                                                              3   48.7                                                                             48.7                                                                              1.5 49.8                                  (3.6)  (3.4)                                                                            (5.6)                                                                             (1)                                                                              (5.3)                                                                             (5.3)                                                                             (2.5)                                                                             (6.5)                                                                            (6.8)                                                                             (1.9)                                                                             (5.6)                                 Mean percentage areas                                                         45.5   54.0                                                                             40.3                                                                              1.3                                                                              56.0                                                                              45.8                                                                              2.2 52.0                                                                             38.7.sup.b                                                                        2.5 60.8.sup.bc                           (4.5)  (5.0)                                                                            (2.5)                                                                             (1)                                                                              (6.7)                                                                             (5.2)                                                                             (2.6)                                                                             (6.4)                                                                            (6.2)                                                                             (2.1)                                                                             (5.3)                                 __________________________________________________________________________     FOG = Fast twitch oxidative glycolytic; FG = fast twitch glycolytic; SO =     slow twitch oxidative;                                                        Within one fibre type, with respect to the innervated control muscles;        .sup.a = p < 0.005, .sup.b = p < 0.05 and with respect to the denervated      control muscle, .sup.c = p < 0.005                                            ***p < 0.005 compared with innervated control;  p < 0.05 compared with        denervated control                                                       

significantly smaller in area (p<0.005) than FOG fibres from innervatedcontrols, but they were significantly increased in area (p<0.05)compared to FOG fibres from control denervated muscles.

If it is assumed that in this experiment the innervated and denervatedmuscles had initially had similar protein contents to the controlanimals in experiment 1, then between 3 and 7 days after denervation thecontrol denervated muscles lost about 1 mg of protein and theclenbuterol-treated denervated muscles gained 1.5 mg.

                                      TABLE 2                                     __________________________________________________________________________    (Section 2)                                                                   Experiment 2-The effect of clenbuterol on chronically denervated muscle       Mean values together with 1 SD indicated in brackets                          Control                                                                       (90.4 (7.8)         Clenbuterol                                               Body weight (g)     90.9 (2.9)                                                __________________________________________________________________________    Innervated                                                                              Denervated                                                                              Innervated                                                                              Denervated                                      Control   Control   Clenbuterol                                                                             Clenbuterol                                     Muscle wet weights (mg)                                                                 ***       *         ***                                             46 (4)    21 (2)    51 (1)    34 (1)                                          Muscle protein (mg/g body weight)                                                       ***       *                                                         0.080 (0.007)                                                                           0.031 (0.005)                                                                           0.089 (0.002)                                                                           0.047 (0.005)                                   Mean cross-sectional areas (sq μm)                                         FOG FG SO FOG FG SO FOG FG SO FOG FG SO                                       915    1124                                                                             354.sup.a                                                                            503.sup.a                                                                        1162.sup.b                                                                           1319                                                                             540.sup.ac                                                                           974.sup.c                                (116)  (68)                                                                             (16)   (47)                                                                             (188)  (247)                                                                            (89)   (93)                                     Mean percentage frequencies                                                   47.1   52.9                                                                             47.8   52.2                                                                             46.8   53.2                                                                             49.8   50.2                                     (1.0)  (1.0)                                                                            (3.7)  (3.7)                                                                            (4.4)  (4.4)                                                                            (5.8)  (5.8)                                    Mean percentage areas                                                         41.9   58.1                                                                             39.1    60.9                                                                            43.8   56.2                                                                             35.5   64.5                                     (3.0)  (3.0)                                                                            (2.0)  (2.0)                                                                            (5.5)  (5.5)                                                                            (6.5)  (6.5)                                    __________________________________________________________________________     FOG = Fast twitch oxidative glycolytic; FG = fast twitch glycolytic; SO =     slow twitch oxidative;                                                        Within one fibre type, with respect to the innervated control                 muscles;.sup.a = p < 0.005, .sup.b = p < 0.05 and with respect to the         denervated control muscle, .sup.c = p < 0.005                                 *p < 0.05, ***p < 0.005 compared with innervated control;    p < 0.005        compared with denervated control                                         

Experiments 1 and 2

In both experiments the cross-sectional area of FOG fibres from theinnervated control muscles were observed to be greater than recorded inprevious experiments. The reason for this difference is not clear;however, it is possible that denervation had imposed some posturalconstraints in the hind limb muscles which gave rise to this differencein fibre size.

DISCUSSION

The results from both experiments indicate that the beta-agonistclenbuterol will not only inhibit but also partially reversedenervation-induced atrophy of rat soleus muscles. The changes in muscleprotein content after denervation suggest that, in the absence ofclenbuterol, denervation results in an initial cessation of musclegrowth but that this may then progress to a true, rather than a relativeatrophy.

In experiment 1 muscle growth was blocked or severely retarded bydenervation. It was not clear whether clenbuterol had inhibited areduction in growth or whether it superimposed a growth stimulus upon adenervation-induced drive towards atrophy. In experiment 2, however, theatrophic response had been well established before the animals receivedthe drug and here clenbuterol led to net protein deposition accompaniedby an increase in fibre size in muscles that would otherwise have beenatrophying.

Denervation is accompanied by an increase in the rate of proteindegradation which is thought to amount largely for the reduction inmuscle protein mass. It is possible that clenbuterol mediates effects onmuscle by reducing protein degradation. The effect of clenbuterol on thegrowth response in chronically denervated muscle accords with such amechanism.

Common to both experiments was the apparently greater sensitivity, indenervated muscle, of the SO fibres to clenbuterol treatment. The basisfor this difference in sensitivity is unknown. Muscles in which SOfibres predominate have a higher fractional rate of protein turnoverthan muscles comprising mainly fast twitch fibres. It may be speculatedthat this is also true of SO fibres in comparison to other fibre typeswithin a single muscle. If so, than an inhibition of protein degradationin a fibre with an inherently more rapid rate of protein degradation maylead to a greater response. This concept can be extended to the effectsof clenbuterol on denervated muscles with their enhanced degradationrates where the proportional effects of clenbuterol on protein mass andSO fibre area were also considerably greater. Thus in experiment 2, thedrug increased the protein content of innervated muscles by 12%, and ofdenervated muscles by 50%. The proportional effect on SO fibre areas wasalso measurably different in innervated and denervated muscles with theincrease of SO fibre area in the former being 17% but 94% in the latter.

It appears then that the process which, in normal muscle, responds toclenbuterol may become more sensitive to the drug in denervated muscleswith similar controlling factors in the two states. Understanding themechanisms of action of clenbuterol may tend to a better understandingof the controls of protein turnover and growth.

Section 3 Propranolol Apparently Separates the Physical andCompositional Characteristics of Muscle Growth Induced by ClenbuterolSummary

The effect of propranolol on clenbuterol-induced changes in muscle fibresize and protein content were studied. Propranolol did not inhibit theability of clenbuterol to stimulate protein accretion but reduced theincrease in muscle fibre size. The compositional and physicalcharacteristics of clenbuterol-induced muscle growth thus appeared to beseparated by propranolol.

INTRODUCTION

The beta-adrenergic agonist, clenbuterol, has been shown to specificallypromote body protein gain and to reduce body fat along withintramuscular fat. Studies in young male rats have shown that theincrease in body protein is apparently confined to cardiac and skeletalmuscle. In sheep the drug had an equally rapid effect on both nitrogenretention, heart rate and body temperature. However, the longer durationand greater drug sensitivity of the nitrogen retention responsesuggested that the two responses might have different mechanistic bases.Using a variety of adrenergic antagonists in rats, it has been foundthat the effects of clenbuterol on lipolysis, heart mass and energyexpenditure were separable from the skeletal muscle growth response. Thebeta-antagonist propranolol blocked the rise in cardiac muscle mass andsignificantly diminished the change in body fat, but had no effect onthe gross skeletal muscle growth response.

We have demonstrated in Section 1 above that the clenbuterol-inducedincrease in muscle protein content is expressed as muscle fibrehypertrophy. In this Section we report the effect of propranolol on thetwo parts of the clenbuterol-induced growth response, namely, musclefibre size and total muscle protein content.

Material and Methods

Male Hooded Lister rats of the Rowett strain were weaned at 19 days ofage and were housed in four groups of 6 animals of equal means bodyweight. The animals were fed to appetite a standard laboratory rat chow(Labsure CRM nuts, K and K Creff, Sussex, UK) and water was freelyavailable at all times. After four days the animals were re-weighed and,if necessary, re-grouped so that all groups had the same mean bodyweight. The animals were housed singly in plastic cages with wirebottoms and fed to appetite the semi-synthetic powdered diet PW3 for afurther 4 days. At this time one group was maintained on PW3 while theother groups were offered either PW3 containing clenbuterol (2 mg/kgdiet), or PW3 containing propranolol (200 mg/kg diet) or PW3 containingboth clenbuterol and propranolol at the doses stated above. All groupswere fed their respective diet for 7 days and their weight and foodintake was recorded daily.

At the end of the experimental period the animals were killed bycervical dislocation and soleus muscles removed and weighted. A smallsample was removed from the mid-belly of each muscle and prepared forhistochemical examination as described in Section 1. Determination offibre type composition was based on the assessment of the stainingreaction for Ca(2+)-activated myofibrillar ATPase at pH 9.4 aftermethanol-free formalin fixation. Those fibres which gave the most densereaction product were designated fast twitch oxidative glycolytic (FOG)fibres, those which gave no reaction product were designated slow twitchoxidative (SO) fibres, and those which gave an intermediate reactionproduct were designated fast twitch glycolytic (FG) fibres. Quantitativeassessments of transverse section stained for ATPase were made fromphotomicrographs using a Hipad digitising tablet (Bausch and Lomb,Austin, Tex.) linked to a Prime 550 computer (Prime Computers Inc.,Framingham, Mass.) programmed to calculate the required parameters fromthe areas delineated on the tablet.

The remainder of the muscle was frozen and stored at -20° C. until usedfor estimations of RNA and protein. The methods used were the same asoutlined above in Section 1.

RESULTS

All the animals grew well on their respective diets and there was littledifference between the mean body weights of each group at the end of theexperiment (control 91.4±2.8 g, clenbuterol 94.6±4.6 g, propranolol89.6±3.5 g, clenbuterol+propranolol 95.7±3.6 g; mean values±SEM n=6 foreach group). Apart from a very slight reduction in food intake on thefirst day of experimental diets, the means daily intakes for each groupwere not significantly different (control 10.1±0.2 g, clenbuterol9.4±0.8 g, propranolol 9.8±0.3 g, clenbuterol+propranolol 10.1±0.2 g:mean values±SEM n=6 for each group). Thus the approximate daily intakeof the drug for each group was either 0.02 mg clenbuterol or 2 mgpropranolol for the single drug diets or the sum of these two for thecombination diet.

Muscle Protein and RNA Content (Table 1)

Dietary administration of clenbuterol gave rise to an increase in muscleweight which was accompanied by a statistically significant increase inboth total protein and RNA. The addition of propranolol alone to thediet caused no change in muscle weight, total protein or RNA content.When clenbuterol and propranolol were added in combination to the diet,a significant increase in total protein content of the muscle wasobserved. Although the total RNA content also increased, the standarddeviation of the mean was such that statistical significance was notachieved. Thus the addition of the beta-antagonist propranolol to thediet did not qualitatively alter the clenbuterol-induced growth responsein rat soleus muscle. In fact the results showed thatclenbuterol+propranolol caused an increase in muscle proteinconcentration.

                                      TABLE 1                                     __________________________________________________________________________    (Section 3)                                                                   The effect of propranolol on the clenbuterol-induced                          changes in protein and RNA in rat soleus muscle                                      Control                                                                              Clen     Prop   Clen + Prop                                     __________________________________________________________________________    Muscle Wt                                                                            45 (4.8)                                                                             54 (4.8) 45 (7.3)                                                                             51 (4.8)                                        (mg)                                                                          Protein                                                                              169                                                                              (13.5)                                                                            174                                                                              (23.8)                                                                              163                                                                              (14.2)                                                                            191                                                                              (29.9)                                       (mg/g)                                                                        Total Prot                                                                           7.6                                                                              (0.8)                                                                             9.3                                                                              (0.8)***                                                                            7.4                                                                              (1.3)                                                                             9.6                                                                              (1.3)**                                      (mg)                                                                          RNA (μg/g)                                                                        2.5                                                                              (0.5)                                                                             2.7                                                                              (0.5) 2.4                                                                              (0.5)                                                                             2.8                                                                              (0.7)                                        Total  113                                                                              (21.8)                                                                            142                                                                              (20.1)*                                                                             107                                                                              (21.8)                                                                            142                                                                              (30.9)                                       RNA (μg)                                                                   RNA/Prot ×                                                                     15.0                                                                             (2.5)                                                                             15.3                                                                             (2.3) 14.1                                                                             (2.8)                                                                             15.0                                                                             (1.9)                                        10.sup.-3                                                                     __________________________________________________________________________

Data are presented as mean values with their standard deviations. Levelsof significance determined with two-tailed Student's t test using themean within-group standard deviations as calculated by a one-wayanalysis of variance; ***p<0.005, **p<0.01, *p<0.05 compared withcontrol.

Muscle Fibre Area and Frequency (Table 2)

Characteristically the addition of clenbuterol to the diet of young malerats produced a skeletal muscle growth response, such that after 7 daysthe drug elicited fibre hypertrophy in the soleus muscles studied.Typically both FOG and SO fibre types showed a significant increase infibre area, which together with the trend towards changes in % frequencyof these two fibre types, resulted in a significant increase in FOG %area and a significant decrease in SO % area.

The muscle from rats fed a diet containing propranolol were very similarto those from control fed animals. However the addition of propranololto the clenbuterol-containing diet resulted in a reduction of theexpected clenbuterol-induced growth response. Fibre areas andfrequencies measured in muscles from animals fed the combination dietdid not differ significantly from those of control muscles.

                  TABLE 2                                                         ______________________________________                                        (Section 3)                                                                   The effect of propranolol on the clenbuterol-induced                          change in fibre area and frequency in rat soleus muscle                       Control      Clen       Prop      Clen + Prop                                 ______________________________________                                        Area (sq. μm)                                                              FOG    939    (154)  1248 (344)*                                                                              919  (99) 1093 (279)                          FG     948    (84)   1017 (308) 864  (41) 987  (146)                          SO     1256   (156)  1568 (398)*                                                                              1257 (164)                                                                              1350 (218)                          % Freq                                                                        FOG    37.7   (8.9)  44.4 (8.6) 39.5 (5.0)                                                                              39.3 (5.6)                          FG     2.0    (2.3)  1.1  (1.1) 0.6  (1.1)                                                                              1.7  (1.5)                          SO     60.3   (6.9)  54.5 (8.2) 59.8 (5.0)                                                                              59.0 (5.6)                          % Area                                                                        FOG    31.3   (7.2)  39.0 (7.1)*                                                                              32.5 (3.7)                                                                              34.3 (5.2)                          FG     1.7    (2.0)  0.9  (0.9) 0.5  (1.0)                                                                              1.3  (1.1)                          SO     67.1   (5.6)  60.1 (6.9)*                                                                              67.0 (3.6)                                                                              64.4 (5.2)                          ______________________________________                                         Data are presented as mean values with their standard deviations.             Fibre type nomenclature                                                       FOG = Fast twitch oxidative glycolytic                                        FG = Fast twitch glycolytic                                                   SO = Slow twitch oxidative                                               

Levels of significance determined with two-tailed Student's t test usingthe mean within-group standard deviations as calculated by a one-wayanalysis of variance; *=p<0.05, compared with control.

DISCUSSION

The effects of the growth-promoting beta-agonists, exemplified byclenbuterol, have proved interesting in a number of respects.Clenbuterol appears to be able to stimulate a muscle-specific increasein protein deposition even in rapidly growing young male rats receivinghigh energy/high protein diets. Similar changes occur in denervatedmuscles as indicated in Section 2 above in which other agents haveproved ineffective. These drugs therefore seem to circumvent aphysiological constraint on muscle growth. This argument can be extendedto changes in protein metabolism in that clenbuterol selectivelysuppresses protein degradation whereas most physiological stimuli tomuscle growth simultaneously increase muscle protein synthesis anddegradation.

The present results suggest that, in the short term at least, it ispossible to separate the accretion of muscle protein from the normallyassociated increase in muscle fibre dimensions. Thus, in confirmation ofprevious results the presence of the mixed-beta antagonist propranololdid not inhibit the ability of clenbuterol to stimulate muscle proteinaccretion but reduced the increase in muscle fibre area that had beenfound to accompany this effect of clenbuterol treatment in previousexperiments. (See Section 1 above).

It should be stressed that these are short-term experiments and that theresults should not be taken to refute the ultimate correlation of musclegrowth, in dimensional terms, with the accretion of muscle protein.Nevertheless, they suggest that somewhat different factors may beinvolved in the control of these two aspects of growth.

The separation of changes in the volume (on the assumption that musclefibre length was the same in clenbuterol and clenbuterol+propranololtreated animals) and the changes in protein content is most simplyexplained by proposing that changes in muscle water had occurred.Clenbuterol alone does not increase the concentration of muscle proteinbut the combination of clenbuterol with the beta-blocker led to a 10%increase in muscle protein concentration. The results of a recentexperiment support this proposition in showing that the waterconcentration was significantly (P<0.025) lower in the muscles of ratsthat had been treated with both clenbuterol and propranolol than inthose of rats that had received each drug individually (clenbuterol73.8±0.3%; propranolol, 73.3±0.3%, clenbuterol+propranolol, 71.6±0.2%mean valves ±SEM n=6 for each group).

Skeletal muscle is both a highly ordered and a heterogeneous tissue inthe sense that it contains intracellular structures, such as complexsarcoplasmic and transverse tubular membrane systems and mitochondria,surrounding a highly structured myofilament array. Evidence hassuggested that "intracellular" water in general and muscle water inparticular does not behave as a freely mobile and exchangeablepopulation of molecules and especially that complex interactions occurbetween f-actin and water. In addition the extracelluar space ofskeletal muscle is constrained both by the fibrillar nature of thetissue and by the presence of the extrafilamental collagenous sheath.Consequently the relationship between the accretion and the two maincomponents of muscle mass, protein and water, may not be as simple asthat in less regularly structured tissues.

A further factor should be considered if changes in the relationshipbetween protein and water accretion underlie the present results. In ourearlier studies of the effects of propranolol on clenbuterol-stimulatedgrowth, it was noted that while the beta-antagonist did not impair theability of clenbuterol to stimulate muscle growth, it significantlyreduced the effects of the beta-agonist on body fat and, perhaps moreimportantly, on total energy expenditure. One factor contributing tothis increase in energy expenditure might be a beta-mediated activationof Na/K ATPase. As the activity of this enzyme is critical to thecontrol of cellular K+ and Na+ it is probably involved in the control ofwater movements across the cell membrane. As a result, while clenbuterolcontinues to promote the deposition of protein in the presence ofpropranolol, the clenbuterol-induced increase in ion pumping may beblocked and the free movement of water inhibited. Whatever theunderlying mechanism the changes that we have observed may be furtherindication that although some of the effects of clenbuterol are trulybetamediated, the particular effects of this drug on muscle proteinaccretion bear little if any relationship to its action as abeta-agonist.

Section 4 The Effect of the Anabolic Agent, Clenbuterol, on OverloadedRat Skeletal Muscle Summary

The dietary administration of clenbuterol to young male rats has beenshown to produce a muscle specific hypertrophic growth response. Thissection demonstrates that the combined effect of drug treatment andhypertrophic stimulus induced by tenotomy produced an additive effect onmuscle growth. This effect was demonstrated in terms of both musclecomposition (protein and RNA) and fibre size.

INTRODUCTION

The protein anabolic effects of the beta adrenergic agonist,clenbuterol, have aroused considered interest over the last few years.Remarkably, the drug appears to have a muscle specific action expressedas muscle fibre hypertrophy as opposed to hyperplasia (see Section 1above).

In young rats the anabolic effect of clenbuterol appears to last forabout a week and it is possible that this is because the increase inmuscle size had reached a critical maximum at this stage. It was ofinterest, therefore, to establish whether the drug could elicit afurther growth response in a muscle that had already been subjected to aseparate hypertrophic stimulus. Tenotomy of gastrocnemius muscles inrats imposes an overload on the remaining functional synergists, ie thesoleus and plantaris muscles which then undergo compensatoryhypertrophy.

The antagonists hold the functionally overloaded muscles in a stretchedstate from which the hypertrophy ensues.

This section describes the effectiveness of the growth promoting drug,clenbuterol, in superimposing further growth on a muscle alreadysubjected to a hypertrophic stimulus induced by tenotomy.

MATERIALS AND METHODS

Male Weanling Hooded Lister rats from the Rowett strain were used andhoused, grouped and fed as described previously (Section 1 above). Inbrief, the animals were divided into two groups of 18 animals of equalmean body weight. The animals were first accustomed to standard rat chowfor 4 days and then to the semi-synthetic powdered diet PW3 for afurther 4 days.

The tenotomy procedure was carried out under ether anaesthesia andaseptic conditions. A small incision was made in the skin at the heeland the tendons of the gastrocnemius were identified and cut.Immediately on recovery from the operative procedure, one group wasoffered the control diet, PW3, whilst the remaining group was offeredPW3 containing clenbuterol (2 mg/kg). From each group (control andclenbuterol), 6 animals were killed after 3, 7 or 11 days and the soleusmuscles were removed from both control and tenotomised limbs. Themuscles were weighed and a small sample was removed from the belly ofthe muscle and prepared for histochemical examination as described inSection 2 above. Determination of fibre type composition was based onthe Ca(2+) ATPase stain and allowed the resolution of slow twitchoxidative (SO), fast twitch oxidative glycolytic (FOG) and fast twitchglycolytic (FG) fibres. Quantitative assessment of fibre areas were madeas described in Section 2 above from photomicrographs of transversesections stained for ATPase activity.

The remainder of the muscle was frozen rapidly and stored at -20° C.until used for determinations of muscle protein and RNA content usingknown methods.

The muscles from the four types of treatment were designated as follows:

    ______________________________________                                        C    soleus from the untentomised limb of control fed rats.                   CT   soleus from the tenotomised limb of control fed rats                     A    soleus from the untentomised limb of clenbuterol (agonist)                    fed rats.                                                                AT   soleus from the tenotomised limb of clenbuterol (agonist)                     fed rats.                                                                ______________________________________                                    

RESULTS

Muscle Protein and RNA Content (Table 1)

In control-fed tenotomy was associated with the typical hypertrophicresponse in the intact synergist muscle, soleus. Muscle weight showed astatistically significant increase within 3 days after surgery, and thiswas accompanied by increases in muscle protein and RNA content (Table1). Over 11 days work-induced hypertrophy increased the RNA content ofgroup CT to a significantly higher value (p<0.005) than in group Amuscles. Tenotomy also resulted in a significant increase (p<0.005) inRNA/protein ratio in the soleus muscles undergoing compensatoryhypertrophy (Table 1).

The group AT muscles showed that the effects of the drug were additiveto those induced by overload. The values for protein and RNA contents ingroup AT muscles were significantly greater than in group A at both 7and 11 days (Table 1).

All muscles exhibited an increase in RNA content as a result of eithertenotomy, clenbuterol administration, or both, and this preceded theincrease in protein gain. The effect was, however, most marked inmuscles subjected to the combined treatment (group AT). Part of theclenbuterol response (group A) involved an increase in RNA, a furtherincrease being achieved in an additive manner by the effect of tenotomy(group AT). Interestingly the CT group muscles showed the smallestincrease in both total protein and RNA between 3 and 7 days, whereas incontrast, clenbuterol treated groups showed large increases in theseparameters over this period.

As observed in muscles from the CT group, there was a statisticallysignificant increase in RNA/protein ratio in muscles from the AT groupfrom 3 days. There was no significant difference between the RNA/proteinratios in muscles from the tenotomised limbs from either dietary groupsafter the initial 3 day period, although both control and clenbuteroltreated groups had significantly (p<0.0005) higher translationcapacities than in either of the unoperated groups.

                                      TABLE 1                                     __________________________________________________________________________    (Section 4).                                                                  Effect of compensatory hypertrophy following tenotomy and of clenbuterol      treatment on the protein and RNA content of rat soleus muscle. Data are       presented as means with their standard deviations (n = 6)                     Unoperated limb   Tenotomised limb                                            Group                                                                             C      A      CT        AT                                                __________________________________________________________________________                      Muscle weight (mg)                                          3.sup.d                                                                           35.0                                                                             (4.0)                                                                             38.0                                                                             (3.4)                                                                             44.8                                                                              (7.9)**                                                                             44.5                                                                             (7.9)*                                         7d  46.0                                                                             (4.3)                                                                             58.0                                                                             (6.7)                                                                             54.0                                                                              (18.0)                                                                              74.0                                                                             (7.2)***   .sup.c                              11d 59.0                                                                             (6.5)                                                                             67.0                                                                             (5.3)*                                                                            76.0                                                                              (6.2)***.sup.c                                                                      83.0                                                                             (7.7)***.sup.a                                                   Total protein (mg)                                          3d  6.7                                                                              (0.9)                                                                             7.3                                                                              (0.7)                                                                             7.9 (1.2) 7.7                                                                              (1.4)                                          7d  9.8                                                                              (1.2)                                                                             12.3                                                                             (1.4)                                                                             10.7                                                                              (3.6) 14.0                                                                             (1.9)**                                        11d 10.8                                                                             (0.9)                                                                             12.4                                                                             (0.9)**                                                                           12.5                                                                              (0.9)***                                                                            14.3                                                                             (0.9)***   .sup.a,                                               Total RNA (μg)                                           3d  100                                                                              (13.2)                                                                            121                                                                              (12.0)                                                                            153 (27.6)***.sup.c                                                                     166                                                                              (34.3)***.sup.c                                7d  117                                                                              (12.2)                                                                            149                                                                              (14.4)                                                                            169 (62.6)*                                                                             231                                                                              (31.7)***  .sup.a                              11d 145                                                                              (21.8)                                                                            160                                                                              (10.8)                                                                            200 (22.3)***                                                                           238                                                                              (13.4)***   .sup.a                                               RNA/protein                                                                   (μg/mg)                                                  3d  15.0                                                                             (0.5)                                                                             16.5                                                                             (0.9)*                                                                            19.4                                                                              (0.7)***.sup.a                                                                      21.4                                                                             (1.2)***   .sup.a                              7d  12.0                                                                             (0.9)                                                                             12.1                                                                             (0.5)                                                                             15.6                                                                              (2.9)***                                                                            16.5                                                                             (0.7)***.sup.a                                 11.sup.d                                                                          13.4                                                                             (0.9)                                                                             12.9                                                                             (0.7)                                                                             16.0                                                                              (1.2)***.sup.a                                                                      16.7                                                                             (0.9)***.sup.a                                 __________________________________________________________________________     Significance values estimated with a twotailed Student's t test using mea     withingroup standard deviation calculated by a oneway analysis of             variance.                                                                     ***p < 0.005; **p < 0.01; *p < 0.05 compared to values from muscles from      unoperated limbs of control fed animals.                                         p < 0.005;   p < 0.01;  p < 0.05 compared to values from muscles from      tenotomised limbs of control fed animals.                                     .sup.a p < 0.005; .sup.b p < 0.01; .sup.c p< 0.05 compared to values from     muscles from unoperated limbs of clenbuterol fed animals                 

MUSCLE FIBRE AREAS (Table 2)

Compensatory muscle fibre hypertrophy associated with tenotomy wasevident in all fibre types in soleus muscles from control fed animalsfrom 7 days onwards, but was statistically significant only at 11 days.The compensatory hypertrophy in group CT muscles was not significantlydifferent from that of clenbuterol treated control muscles (group A;Table 2). The lack of statistically significant hypertrophy in the groupCT and group A muscles at 3 days is unexplained and unexpected in viewof the significant hypertrophy in group AT muscles.

The effects of clenbuterol and tenotomy were (in all cases except 3d)additive, such that the FOG and SO fibre areas in the AT group muscleswere significantly greater than in any other group (p<0.05, 7d and 11d(Table 2)). A statistically significant hypertrophy was evident in themuscles from the AT group prior to that seen in comparable muscle fromcontrol fed animals. This response occurred at a time when thecontralateral muscles from these clenbuterol fed animals were showing asmall stimulation in weight and protein gain (Table 2). In terms ofpercentage change from their respective unoperated controls, however, at11 days, the muscles from the CT group showed the greater percentageincrease in fibre area, in agreement with the changes in weight andprotein.

                                      TABLE 2                                     __________________________________________________________________________    (Section 2):                                                                  Effect of compensatory hypertrophy following tentotomy and of                 clenbuterol                                                                   treatment on fibre area in rat soleus muscles. Data are presented as          means                                                                         with their standard deviations (n = 6)                                        Unoperated limb    Tenotomised limb                                           Mean area (sq μm)                                                                             Mean area (sq μm)                                       FOG     FG  SO     FOG   FG   SO                                              __________________________________________________________________________    3d  737 703 996 3d 593   566  868                                             C   (77)                                                                              (50)                                                                              (38)                                                                              CT (40)  (42) (102)                                           3d  742 628 898 3d 918***                                                                              918  1286**   .sup.a                                 A   (217)                                                                             (153)                                                                             (165)                                                                             AT (163) (192)                                                                              (180)                                           7d  870 777 1172                                                                              7d 970   980  1239                                            C   (146)                                                                             (158)                                                                             (265)                                                                             CT (242) (232)                                                                              (287)                                           7d  1123                                                                              1096*                                                                             1307                                                                              7d 1398***  .sup.c                                                                     1234***                                                                            1672***  .sup.c                                 A   (195)                                                                             (263)                                                                             (159)                                                                             AT (141) (219)                                                                              (283)                                           11d 1066                                                                              895 1256                                                                              11d                                                                              1328* 1130*                                                                              1661***                                         C   (170)                                                                             (96)                                                                              (109)                                                                             CT (141) (219)                                                                              (283)                                           11d 1337                                                                              1153                                                                              1487                                                                              11d                                                                              1537*** .sup.c                                                                      1401*** .sup.c                                                                     1799***.sup.b                                   A   (213)                                                                             (127)                                                                             (166)                                                                             AT (101) (163)                                                                              (118)                                           __________________________________________________________________________     Significance values estimated with a twotailed Student's t test using the     mean withingroup standard deviation as calculated by a oneway analysis of     variance.                                                                     ***p < 0.005; **p < 0.01; *p < 0.05 compared to values from muscles from      unoperated limbs of control fed animals.                                         p < 0.005;   p < 0.01;  p < 0.05 compared to values from muscles from      tenotomised limbs of control fed animals.                                     .sup.a p < 0.005; .sup.b < 0.01; .sup.c p < 0.05 compared to values from      muscles from unoperated limbs of clenbuterol fed animals                 

DISCUSSION

Previous studies have shown that the muscle-specific anabolic effects ofclenbuterol were expressed as muscle fibre hypertrophy (Section 1above). The results of the present experiments demonstrated that whileclenbuterol treatment potentiated muscle growth, the drug does notelicit all the latent growth of which the tissue is capable.

Muscles exposed to either clenbuterol or tenotomy of their synergistsexhibited hypertrophy. The response of such muscles was characterised byan increase in total muscle protein and RNA content together with anincrease in fibre cross-sectional area. By 11 days, both experimentalmanipulations produced a comparable degree of response in terms of totalprotein content and fibre size. Tenotomy-induced compensatoryhypertrophy, however, led to a much more marked increase in RNA/proteinratio than was evident in muscles from the clenbuterol treated group(Table 1). This data would be consistent with the fact that compensatoryhypertrophy is associated with elevated rates of protein synthesis,while clenbuterol has been shown to reduce protein degradation and havelittle effect on the fractional rate of synthesis. This indicated thatwhile the net result of both treatments was similar, hypertrophy, themechanism by which this was achieved might differ between the two.

Consequently it was not surprising that the combination of clenbuteroltreatment with the tenotomy-induced hypertrophic drive ultimately (by 11days) produced a significantly greater response (p<0.005) in terms oftotal muscle protein and RNA than either treatment alone. This responseto the combined treatments was also broadly evident in the fibre sizes.The fact that the SO fibres in the muscles from the tenotomised limbwere not significantly different between treatment groups may beimportant. From this, it can be inferred that these oxidative fibres hadreached a maximum size beyond which diffusion of oxygen, sufficient forthe particular metabolism of these oxidative muscles, might becomelimiting. The FOG and FG fibre types being capable of anaerobicmetabolism would not be constrained to the same extent. This being so,it is possible that with the combination of the two treatments themaximum growth potential under these particular circumstances may havebeen realised.

Section 5 The Effect of Beta-agonists and Beta-antagonists on MuscleGrowth and Body Composition of Young Rats (RATTUS SP.) Summary

1. The addition of the beta-selective adrenergic agonist clenbuterol tothe diet was associated with an increase in the protein and RNA ofskeletal and cardiac muscle, a reduction in fat deposition and anincrease in energy expenditure.

2. Neither propranolol nor atenolol blocked the effect of clenbuterol onmuscle mass but both reduced its effect on cardiac and fat mass andenergy expenditure.

3. Five other beta-agonists were tested. All increase the interscapularbrown fat mass and lowered body fat but only two increased skeletalmuscle protein.

4. It is concluded that the anabolic and anti-lipogenic actions ofcertain beta-agonists are mechanistically distinct.

INTRODUCTION

Some but not all beta-selective adrenergic agonists stimulate thedeposition of body protein and inhibit that of body fat. Their effectshave been observed in a number of species, they are effective inuncastrated male animals and, most strikingly, their anabolic propertiesappear to be confined to skeletal and cardiac muscle. Some results havesuggested that the increase in muscle protein accretion arises from amarkedly lower rate of muscle protein degradation.

It has been found that in sheep, one of these drugs, clenbuterol,administered as an abomasal infusion, increased nitrogen retentionwithin three hours and that the increase in nitrogen retention coincidedwith an increase in both heart rate and body temperature. However thecardiovascular response was of short (c. 30h) duration while nitrogenretention remained elevated for 6 weeks. The differences in thechronicities of the nitrogen retention and cardiovascular responses aswell as differences in their dose sensitivities suggest that they arisefrom different mechanisms. In particular it might be questioned whether,if clenbuterol directly promotes muscle growth, its action in thisrespect is related to beta-stimulation.

In this Section we report investigations of the sensitivity tobeta-antagonists of the stimulation of muscle growth and the inhibitionof fat deposition by clenbuterol and of the effect of some otherbeta-agonists on tissue growth and fat deposition.

MATERIALS AND METHODS

Materials

With the exception of Clenbuterol, which was a gift from BoehringerIngelheim, the various drugs were obtained commercially. Materials forthe assays were obtained either from Sigma Chemical Co. (Poole, Dorset,UK) or from B.D.H. PLC (Poole, Dorset, UK).

Animals and Feeding

The animals were Hood Lister rats of the inbred Rowett strain. Theanimals had been bred in a specific pathogen free unit. After weaning at19 d post partum the animals were housed in a minimal disease animalhouse maintained at 23° C. with a 12 hour light/dark cycle (lights on at07.00 h). Male rats were used in experiment 1 and female rats inexperiment 2. Previous work has shown that the two sexes do not differin their responses to Clenbuterol.

After weaning the animals were housed in groups of 6 of equal mean bodyweight and for the next four days they were offered, to appetite, astandard laboratory diet (Labsure, K & K Greef, Croydon, Surrey, UK).They were then reweighed and if necessary regrouped so that each grouphad the same mean body weight at the start of the experimental periodand hence had the same weight gain during the preliminary period. Atthis time any animals that differed from the mean by more than 2standard deviations (±3 g) were excluded. In all 12 animals from thestarting 168 were excluded on this basis.

The animals were then housed singly in clear plastic cages provided withslatted floors and allowed free access to a powdered diet (PW3) for afurther 4 days after which they either continued to receive this diet orPW3 containing various drugs. The drugs were Clenbuterol, Isoetharine,Orciprenaline, Reproterol, Salbutamol, and Terbutaline. The powdereddrugs were mixed in a small quantity of diet PW3 and appropriate amountsadded to the main diet formulation. The diet was then mixed for at least30 minutes at 200 revolutions per minute in a domestic food mixer usingthe beater attachment.

From this time their body weights (measured between 09:30 and 10:30) andfood intakes (corrected for spillage) were recorded daily. Groups ofanimals were killed at the start of the experiment and after 7 and 21 din experiment 1. In experiment 2 the animals were killed after 15 d.

Slaughter and Dissection

The animals were taken from the animal house at 09:00 h. They werekilled by cervical dislocation no later than 3 h after transfer to thelaboratory and were weighed immediately after death. The wholegastro-intestinal tract, the liver, both kidneys and the heart wereremoved. The organs were thoroughly blotted before weighing and theheart was exsanguinated. The hind limbs were then severed from the body,skinned and fixed to the bench by the foot. The achilles tendon wassevered and the gastrocnemius/plantaris/soleus muscle group peeledproximally. The muscles were separated, each pair was pooled, placed ina tared polythene bag, weighed and frozen in liquid nitrogen. Thetissues were stored in sealed bags at -20° C. until analysed within amonth of dissection. The carcase was then weighed and frozen at -20° C.

Muscle Composition

The muscle samples were frozen in liquid nitrogen and powdered between 2aluminium blocks that had been precooled on solid CO₂. Aliquots(approximately 100 mg) of the powder were weighed accurately andhomogenised in 3 ml of 0.5M Perchloric acid (PCA). The homogenate wascentrifuged at 6000×g for 15 min and the pellet was resuspended in 2 ml0.5M PCA. After centrifugation the two supernatants were pooled andstored frozen for subsequent measurement of muscle creatine content. Thepellet was incubated in 10 ml 0.3M NaOH for 1 h at 37° C. and a 1 mlaliquot of the solution taken for protein measurement. The remainder wasmixed with 1 ml 3M PCA, cooled to 4° C. for 30 min and centrifuged. Thesupernatant was taken for estimation of the RNA content from the ratioof optical density (OD) at 260 nm and 232 nm. RNA was calculated fromthe formula:

    RNA(micro-g/ml)=10.34 (3.17×OD 260-0.75×OD 232)

In some experiments the pellet was then resuspended in 10 ml 0.5 ml PCA,heated for 30 min at 90° C., centrifuged and the DNA content of thesupernatant estimated by a known method. All the muscles showed similarresponses and therefore, for the sake of simplicity, only the resultsobtained in the gastrocnemius muscles are shown below.

Gross Body Composition

The frozen carcase was cut into approximately 3 cm cubes and lyophilisedfor 4 d, the last 2 days of which were at 40° C. The dried samples werepowdered in a Braun Multimix food processor and triplicate samples weretaken for analysis of nitrogen and fat. Body protein was calculated asN×62.5 and total body energy content was calculated as:

    Energy (kj)=(Body N(g)×148.1)+(Body fat (g)×39.6)

Estimation of Muscle Mass

The measurement of total muscle mass was based on the assumption thatthe body pool of creatine represents almost entirely the creatine inskeletal muscle. By measuring the body content of creatine and relatingthis to the ratio of creatine:protein in muscle samples from the sameanimal an estimate of the total muscle protein pool can be obtained.

Duplicate samples of the dry powdered carcase (approximately 300 mg)were homogenised in 3 ml 0.5M PCA and then centrifuged at 6000×g for 20min. The pellet was washed with a further 2 ml of PCA. The supernatantswere combined and neutralised (pH 4-6) with a known volume of 4M KOH.After centrifugation the supernatant was re-acidified with 0.25 ml of5.4M HCl and the solution was autoclaved for 30 min at 15 lbs/sq in. inorder to convert creatine to creatinine. The creatinine concentrationwas measured against standards prepared from creatine treated in anidentical manner. The creatine content of the PCA supernatants ofgastrocnemius muscle homogenates was also estimated. Previous work inrabbits has shown that the method gives results that areindistinguishable from those obtained by total dissection.

Statistics

The significance of differences between means was assessed by two-tailedt-tests. In experiment 2 changes in body fat, hepatic, cardiac and totalmuscle mass were related to one another by the Spearmann RankCorrelation test. A value of P (two-tailed) less than 0.05 was taken asbeing statistically significant.

RESULTS Experiment 1.

The effects of beta-antagonists on clenbuterol action

Although the presence of clenbuterol in the diet had no effect on foodintake it was associated with an increase in the protein and RNA (butnot the DNA) contents of the gastrocnemius muscle (Table 2) and anincrease in the total muscle protein mass (Table 3). Rather thandecreasing this effect of clenbuterol both antagonists produced afurther small, but statistically significant increase in gastrocnemiusprotein content. Cardiac protein, RNA and DNA were also higher inanimals that had received clenbuterol alone. However both propranololand atenolol significantly reduced the effect of clenbuterol on theprotein and RNA content of the heart.

Body protein, fat and calculated energy expenditure

Four observations were noteworthy (Table 3). First, the effect ofclenbuterol on both total body and total muscle protein associated withclenbuterol treatment was largely complete by 7 d of treatment. Second,the increase in total body protein was entirely accounted for by theincrease in muscle protein mass. Third, neither of the antagonistsimpaired the effect of clenbuterol on total muscle and total bodyprotein deposition and again there was a tendency (although notsignificant) for total muscle protein to be higher in groups CP, CPIIand CA. Fourth, the lower rate of fat deposition associated with theprolonged ingestion of clenbuterol was partially reversed by bothpropranolol and atenolol. It should be noted that this effect was notcompletely revered, and the body fat mass of groups CP, CPII and CA wasstill significantly lower than that of the control animals.

When based on the difference between metabolizable energy intake andbody energy gain it appeared that clenbuterol increased total energyexpenditure by about 8% (P<0.001) and that both antagonists completelyreversed this effect of the beta-agonist.

Experiment 2.

The effects of beta(2)-agonists on growth and body composition

Of the agonists studied only clenbuterol markedly increased the mass ofthe leg muscles (Table 4). Although orciprenaline and isoetharine bothproduced some effect this was less than that associated with clenbuteroltreatment. Isoetharine and salbutamol increased cardiac masssignificantly and terbutaline and reproterol reduced the hepatic mass.With the exception of orciprenaline all the drugs increased the mass ofthe interscapular brown fat pads.

Orciprenaline and isoetharine increased body protein mass (+5% P<0.05)although not as effectively as clenbuterol (+11% P<0,001). Terbutalineand orciprenaline also reduced body fat mass (-14%) but again neitherdrug had as marked an effect as clenbuterol (-26%). The Spearmann RankCorrelation test revealed a significant relationship between thedecreases in body fat and hepatic mass (R=0.421: P<0.05). The change inleg muscle mass correlated significantly with the increase in bodyprotein but with no other measurement.

DISCUSSION

Although a lower rate of fat deposition has been a common finding inanimals treated with a variety of both alpha and beta selectiveadrenergic agonists, only a few beta-agonists increase body protein.Therefore it might be questionea whether the apparently interrelatedeffects of clenbuterol, fenoterol and cimaterol on body protein and fatshare a common, beta-mediated mechanism.

The present results in clenbuterol treated animals confirmed ourprevious findings that the growth promotion appeared to be confined toskeletal muscle protein and RNA were not accompanied by increases in DNAsuggesting that the growth response was hypertrophic rather thanhyperplastic in nature. This has been confirmed by measurements of fibrenumber and diameter as shown in Section 1 above. In heart, however,there was an increase in DNA content.

The transient nature of the effect of clenbuterol on muscle growth mightbe interpreted as indicating a tachyphylaxis. However the drug had aprogressive effect on cardiac protein (increased by 14 mg at 7 d and by35 mg at 21 d) and on fat mass (reduced by 2.5 g at 7 d and by 8.7 g at21 d) and this suggests that tachyphylaxis is not the explanation. It ispossible that the cessation of accelerated muscle growth results from aneventual limitation imposed by an unaltered rate of skeletal growth.Sheep and cattle, in which the effect of clenbuterol on body nitrogengain is much more prolonged, have a much lower weight-specific growthrate.

Neither the mixed-beta antagonist propranolol nor the beta(1)-selectiveantagonist atenolol blocked the effect of clenbuterol on muscle growth.Furthermore in preliminary work with a beta(2)-antagonist--Hoffman LaRoche compound Ro 22-4574--we were also unable to find any inhibitoryaction on clenbuterol-stimulated muscle growth. In fact thebeta-antagonists tended to produce a further small increase in muscleprotein deposition. It is possible that the effects of clenbuterol onbody fat, intramuscular fat, and muscle glycogen might place somelimitation on muscle growth. By reducing these "deleterious" effects ofclenbuterol the beta-antagonists may have thereby reduced theirinhibitory effects on muscle growth.

These results contrasted with the substantial inhibition by bothantagonists of the effect of clenbuterol on energy expenditure andcardiac growth and their partial inhibition of the effect of clenbuterolon body fat deposition. In sheep we have noted a marked effect ofclenbuterol on heart rate, an effect that coincided with a reduction innitrogen excretion. However these two effects had different dosesensitivities (nitrogen retention being the more sensitive) andchronicities (the increase in heart rate being a transient phenomenon)and these observations suggested that they might not be directlymechanistically related. As both the increase in cardiac growth andtotal energy expenditure associated with clenbuterol treatment wereblocked by the beta(1)-selective antagonist atenolol, it is possiblethat the increase in cardiac growth is a beta(1)-mediated response andthat it results from cardiac overload, rather than from the directaction of clenbuterol as an anabolic agent.

Some degree of caution should be exercised. It remains possible that thedifferential responses of muscle growth on the one hand, and of fatdeposition and cardiac growth on the other merely result fromdifferences in the affinity of the appropriate beta receptors toclenbuterol and propranolol. To investigate this possibility we exposedrats to the high dose of propranolol for 4 days before exposing them toclenbuterol and propranolol. Again the antagonist did not block theeffect of clenbuterol on muscle growth [Gastrocnemius protein content(mg); Control, 288±9; Clenbuterol+Propranolol, 392±12].

The results obtained with the other beta-agonists also demonstrate thecomplex pharmacology of the growth response. The drugs were selectedinitially for differences in their absolute potencies and beta(2)selectivities. Although they were administered at twice the dose ofclenbuterol none were as effective as clenbuterol in promoting musclegrowth. Salbutamol and orciprenaline significantly decreased body fat, 4of the 5 drugs increased the mass of the brown fat pads and all tendedto reduce hepatic mass, an effect that they shared with clenbuterol.Significant Rank Correlations between the effects of the drugs oncardiac, hepatic and fat mass were found but, with the exception of bodyprotein mass, the change in leg muscle mass did not significantlycorrelate with any of the other measurements. Once again therefore theeffects of the drugs on muscle growth and their effects on body fat andcardiac mass were separated.

Although the results cast doubt on a close relationship between betaactivity and the stimulation of muscle growth they offer no explanationas to the mechanisms of the anabolic action. Our previous resultssuggest that clenbuterol increases muscle protein deposition by reducingthe rate of muscle protein degradation. This mechanism is different fromthat underlying the promotion of muscle growth by change in tension,work-load and insulin. Furthermore it is not certain whether the effectof clenbuterol on muscle protein deposition is direct or whether it isrelated to some other hormonal change. It is of interest that thenon-selective beta-agonist isoproteronol reduces protein degradation inisolated muscles and in the hemi-corpus preparation but we have beenunable to demonstrate a similar effect of clenbuterol (over the range10⁻¹¹ M to 10⁻⁸ M) in isolated muscles. Nevertheless clenbuterol atconcentrations in excess of 10⁻¹⁰ M reduced muscle glycogen in vitro. Itis possible that a hitherto unidentified metabolite of clenbuterol isresponsible for the stimulation of muscle growth while changes in fatdeposition, glycogenolysis and energy expenditure are direct,beta-mediated, responses to the drug itself.

                  TABLE 1                                                         ______________________________________                                        (Section 5):                                                                  Designation of the diets offered to the animals                               Diet group                                                                            Addition to the diet                                                  ______________________________________                                        C       None                                                                  CL      Clenbuterol (2 mg/kg)                                                 P       Propranolol (20 mg/kg)                                                PIII    Propranolol (200 mg/kg)                                               CP      Clenbuterol (2 mg/kg) + Propranolol (20 mg/kg)                        CPII    Clenbuterol (2 mg/kg) + Propranolol (200 mg/kg)                       A       Atenol (20 mg/kg)                                                     CA      Clenbuterol (2 mg/kg) + Atenolol (20 mg/kg)                           T       Terbutaline (4 mg/kg)                                                 O       Orciprenaline (4 mg/kg)                                               R       Reproterol (4 mg/kg)                                                  S       Salbutamol (4 mg/kg)                                                  I       Isoetharine (4 mg/kg)                                                 ______________________________________                                    

                                      TABLE 2                                     __________________________________________________________________________    (Section 5)                                                                   The protein, RNA and DNA contents of the gastrocnemius muscles and the        heart of male rats at the end of                                              a 21-day period over which they received a control diet or one of the         diets that are listed in Table 1.                                             Mean values together with one standard error of the mean for the numbers      indicated.                                                                    Gastrocnemius Muscle         Cardiac Muscle                                   Group                                                                             N Protein (mg)                                                                           RNA (μg)                                                                           DNA (μg)                                                                         Protein (mg)                                                                         RNA (μg)                                                                           DNA (μg)                       __________________________________________________________________________    C   18                                                                              282 ±                                                                          5    2340 ±                                                                         60  810 ± 39                                                                         138 ±                                                                          3  1580 ±                                                                         30  1256 ± 20                      P   12                                                                              272 ±                                                                          5    2350 ±                                                                         50  ND    129 ±                                                                          4  1580 ±                                                                         30  ND                                PII 6 290 ±                                                                          5    2340 ±                                                                         90  818 ± 48                                                                         144 ±                                                                          3  1630 ±                                                                         40  1264 ± 30                      A   12                                                                              289 ±                                                                          9    2360 ±                                                                         50  ND    131 ±                                                                          6  1660 ±                                                                         50  ND                                CL  18                                                                              312 ±                                                                          3*** 2540 ±                                                                         50***                                                                             756 ± 47                                                                         164 ±                                                                          5***                                                                             1780 ±                                                                         30***                                                                              1310 ± 20*                    CP  12                                                                              315 ±                                                                          4*** 2490 ±                                                                         40* ND    144 ±                                                                          3  1620 ±                                                                         50  ND                                CPII                                                                              6 320 ±                                                                          3***, @                                                                            2550 ±                                                                         70***                                                                             776 ± 42                                                                         145 ±                                                                          5  1690 ±                                                                         40  1305 ± 30                      CA  12                                                                              338 ±                                                                          8***, @                                                                            2620 ±                                                                         40  ND    141 ±                                                                          4  1700 ±                                                                         30*,                                                                              ND                                __________________________________________________________________________     The statistical significance of differences are indicated:                    Significantly higher than group C, *P < 0.05; ***P < 0.001                    Significantly lower than group CL,  P < 0.05;   P < 0.01                      Significantly higher than group CL, @P < 0.05                            

                                      TABLE 3                                     __________________________________________________________________________    (Section 5)                                                                   Total body and muscle protein,                                                body fat and calculated energy expenditure of young male rats offered         to appetite for 7 or 21 days a control diet or the diets that are             described in Table 1.                                                         Mean values together with one standard error of the mean for the numbers      indicated.                                                                                                       Total Energy                                        Body Protein                                                                          Muscle Protein    Expenditure                                Day                                                                              Group                                                                             N (g)     (g)     Body Fat (g)                                                                            (kj)                                       __________________________________________________________________________    0  IS  9 7.5 ±                                                                          0.2 2.8 ±                                                                          0.1 3.8 ±                                                                          0.3   --                                         7  C   6 15.9 ±                                                                         0.5 7.3 ±                                                                          0.3 10.5 ±                                                                         0.7   855 ±                                                                          28.sup.b                                  P   6 16.3 ±                                                                         0.4 7.7 ±                                                                          0.2 9.9 ±                                                                          0.5   859 ±                                                                          24                                        CL  6 18.0 ±                                                                         0.77***                                                                           9.4 ±                                                                          0.3***                                                                            8.0 ±                                                                          0.5   912 ±                                                                          10*                                       CP  6 17.8 ±                                                                         0.5***                                                                            9.4 ±                                                                          0.5***                                                                            9.4 ±                                                                          0.4@  858 ±                                                                          14                                     21 C   18                                                                              30.5 ±                                                                         0.5 15.2 ±                                                                         0.4 22.6 ±                                                                         1.1   3327 ±                                                                         41.sup.c                                  P   12                                                                              30.9 ±                                                                         0.5 15.7 ±                                                                         0.4 19.8 ±                                                                         0.5   3366 ±                                                                         63                                        PII 6 30.2 ±                                                                         0.6 14.9 ±                                                                         0.4 20.2 ±                                                                         1.8   3310 ±                                                                         112                                       A   12                                                                              31.1 ±                                                                         0.5 16.1 ±                                                                         0.6 21.5 ±                                                                         1.7   3494 ±                                                                         101                                       CL  18                                                                              32.8 ±                                                                         0.5***                                                                            18.0 ±                                                                         0.4***                                                                            13.9 ±                                                                         0.4   3716 ±                                                                         53***                                     CL  12                                                                              33.9 ±                                                                         0.5***                                                                            19.2 ±                                                                         0.7***                                                                            17.8 ±                                                                         0.6  , @@                                                                           3420 ±                                                                         49                                        CPII                                                                              6 32.7 ±                                                                         0.5***                                                                            18.2 ±                                                                         0.6***                                                                            18.2 ±                                                                         0.6  , @@                                                                           3125 ±                                                                         70                                        CA  12                                                                              33.2 ±                                                                         0.5***                                                                            18.2 ±                                                                         0.4***                                                                            17.6 ±                                                                         0.9  , @@                                                                           3385 ±                                                                         55                                     __________________________________________________________________________     .sup.a Animals slaughtered on the day of first exposure to the                experimental diets.                                                           .sup.b Energy expenditure from 0-7 days                                        .sup.c Energy expenditure from 7 to 21 days                                  The statistical significance of the differences are indicated:                Significantly higher than group C, ***P < 0.001                               Significantly lower than group CL,   P < 0.01;    P < 0.001                   Significantly higher than group CL, @P < 0.05; @@P < 0.01                

                                      TABLE 4                                     __________________________________________________________________________    (Section 5)                                                                   Tissue weights (mg), body                                                     protein (g) and body fat (g) of groups of 6 female rats offered to            appetite                                                                      for 15 days control diet or the diets described in Table 1. Mean values       together with a common estimate of variance pooled standard deviation         PSD                                                                           Group                                                                         Tissue                                                                              C   CL   T   R   I   S   O   PSD                                        __________________________________________________________________________    Liver 7370                                                                              6530 6750                                                                              6950                                                                              6980                                                                              7000                                                                              7150                                                                              344                                        Heart 654 818***                                                                             653 669 724*                                                                              729*                                                                              709 48                                         Muscle.sup.a                                                                        1841                                                                              2144***                                                                            1822                                                                              1886                                                                              1921*                                                                             1876                                                                              1960*                                                                             94                                         Brown Fat                                                                           448 524**                                                                              449***                                                                            511*                                                                              524**                                                                             531**                                                                             434 60                                         Body  24.0                                                                              26.7***                                                                            23.9                                                                              24.6                                                                              25.4                                                                              25.1                                                                              25.3*                                                                             1.0                                        Protein                                                                       Body Fat                                                                            11.4                                                                              8.4  9.4 10.0                                                                              10.6                                                                              10.7                                                                              10.2                                                                              1.5                                        __________________________________________________________________________     .sup.a The combined weights of the gastrocnemius, plantaris, soleus and       extensor digitorum longus muscles of both limbs.                              The statistical significance of differences are indicated:                    Significantly higher than group C, *P < 0.05; **P < 0.01; ***P < 0.001        Significantly lower than group C,  P < 0.05;   P < 0.01;    P < 0.001    

Section 6 Clenbuterol, a beta agonist, induces growth in innervated anddenervated rat soleus muscle via apparently different mechanisms Summary

Dietary administration of the anabolic agent clenbuterol has alreadybeen shown to inhibit or reverse denervation-induced atrophy in ratsoleus muscles. We now show that the ameliorative effects of clenbuterolin denervated rat muscles are due principally to a large increase inprotein synthetic capacity and a normalised translational efficiency.The responses of innervated and denervated muscles are thereforefundamentally different, the changes in denervated muscles beingreminiscent of the classical pleiotypic response of cells to growthfactors.

INTRODUCTION

Beta-adrenergic agonists have been proposed for use as thermogenicagents to reduce body fat but a limited number of these drugs have anadditional anabolic action. Drugs such as clenbuterol, fenoterol andcimaterol, that are effective in this respect are remarkable in having amuscle specific action across a wide range of species. The anabolicaction of clenbuterol in innervated rat muscles is expressed as fibrehypertrophy (see Section 1 above) and results primarily from a reductionin protein degradation. We have shown in Section 2 above that indenervated rat soleus muscles undergoing atrophy, due primarily to anelevation in protein degradation, clenbuterol is highly effective atinhibiting or reversing the atrophic response.

This observation suggested that, since the effect of clenbuterolappeared to be mediated by a reduction in degradation, the action of thedrug on the growth of denervated muscles would accord with such amechanism. The present experiment was designed to examine thispossibility by studying the changes in protein turnover in denervatedrat soleus muscles exposed to clenbuterol.

MATERIAL AND METHODS

Animals and Experimental Protocol

Two identical experiments were carried out, one for determination ofprotein turnover, the other for histochemical analysis. For eachexperiment male Hooded Lister rats of the Rowett strain were weaned at19 d post partum and divided into 11 groups of 6 animals of equal meanweight. The initial feeding regime was exactly as described previouslyin Section 1 above. Rats (60±2 g body weight), 6 days post weaning (19d), which had been accustomed to the control diet PW3 for 3 days weresubjected to unilateral removal of 0.7-1.2 cm of sciatic nerve, underether anaesthesia and aseptic conditions. The animals were housed singlyin flat bottomed cages and their body weights recorded daily. At 10 amon day 4 part denervation the rats were offered to appetite control diet(5 groups) or diet containing the beta-adrenergic agonist, clenbuterol(2 mg/kg; 5 groups). One group of rats was analysed as a zero timecontrol and then one group from each of the control and plus-clenbuterolgroups were analysed after 12, 24, 36, 72 and 168 h.

The animals were killed and the soleus muscles from both the innervatedand denervated limbs removed and frozen suitably for the subsequentanalysis (see below).

Determination of the fractional rate of protein synthesis and muscleprotein and RNA contents

The fractional rate of protein synthesis (K_(s)) was determined, using aflooding dose of 150 mM tritium-labelled 2,6-³ H-Phenylalanine (75microCuries/ml; 1 ml per rat), in both the innervated and denervatedsoleus muscles of control and clenbuterol treated rats. Unanaesthetisedrats were immobilised, by wrapping in a J-cloth, and the large dose of ³H-Phenylalanine was injected into a lateral tail vein. After exactly 10minutes the animals were killed and the hind limbs removed. The legswere rapidly skinned and cooled in iced water before the soleus muscleswere dissected and frozen and stored in liquid nitrogen. Muscles werehomogenised and prepared for measurement of the specific radioactivityof free and protein-bound phenylalanine. Total protein synthesis wascalculated as K_(s) x protein content.

Muscle protein and RNA contents were determined by known methods.

The fractional rate of degradation was estimated as the differencebetween K_(s) and the fractional rate of protein gain (k_(g)). Thelatter was estimated from the change in protein content duringsuccessive time points and assumes the rate to have been linear.

Histochemistry

The histochemical analysis of muscle sections was carried out exactly asdescribed in Section 2 above.

Statistics

Data were assessed by analysis of variance procedures using a split plotdesign. Where appropriate, comparisons between effects were made usingthe t-statistic derived from the standard error of the difference(S.E.D.; 36 degrees of freedom).

RESULTS

Sciatic denervation led to a near cessation of soleus growth with themuscle protein mass (Table 1) and fibre areas changing little over theperiod 'studied. The presence of clenbuter in the diet stimulated thegrowth of both the innervated and denervated muscles (Table 1) but, aswe have shown in Section 2 above, denervated muscles showed a relativelygreater proportional improvement in protein mass, 71% for denervatedversus 25% for innervated, when compared to appropriate controls(P<0.001). The nature of the compositional changes induced byclenbuterol was influenced by nerve status. In innervated muscles bothprotein and RNA mass increased in proportion (Table 1), while in thedenervated muscles the RNA/protein ratio was significantly elevated(P<0.01) by day 1 and remained so until day 3 of exposure toclenbuterol.

By 4 d of denervation the classical changes in protein metabolism wereestablished with both total and fractional rates of protein synthesis(K_(s)) depressed (by 60% and 29% respectively) and fractional rate ofdegradation (K_(d)) augmented (Table 2). The fibres in these denervatedmuscles were characteristically small (see Section 2 above).Administration of the drug at this time produced striking differences ineffects on protein metabolism between innervated and denervated muscles(Table 2). In both cases clenbuterol produced a rapid decrease in K_(d)of approximately 50% over the first 24 hours (Table 2) and this wasfollowed by a lesser reduction (15%) for the remaining 6 d (Table 2). Ininnervated muscles the drug did not influence the K_(s) (Table 2), andthe increase in fibre size could be accounted for by the decrease inprotein catabolism. In contrast, denervated muscles exposed toclenbuterol showed a stimulation in the K_(s) and an improvement in thetranslational efficiency (g protein synthesised per g RNA) compared tountreated denervated muscles (Table 2). Hence the increase in fibre sizewas, in part, accounted for by the stimulation in synthesis and in partby the reduction in degradation. After 7 d of clenbuterol administrationthe denervated muscles exhibited compositional and kinetic propertiessimilar to those of normal innervated muscles of that size.

DISCUSSION

The characteristic anabolism in response to clenbuterol was once againobserved in the innervated muscles. In these muscles the increase infibre size and protein gain (Table 1) associated with the drug treatmentcould be accounted for by a decrease in protein catabolism (Table 2). Inthe case of the denervated muscles (Tables 1, 2) only half of theprotein anabolism was achieved through reduced degradation, theremainder resulted from a stimulation in both the fractional (Table 2)and total rate of protein synthesis. This arose from changes in twoprocesses which showed different temporal sequences. First, thetranslational efficiency, which was depressed by 25% as a result ofdenervation, was restored within 12-24 h of starting clenbuteroltreatment to that of control values (Table 2). Second, RNA accretion,and thus total potential translational capacity, was enhanced over theperiod 24-72 h. In consequence of these two effects, an increase between24-36 h of 58% in the fractional rate of protein synthesis was observedcompared with nontreated denervated muscles and the increase was stillsignificant at 72 h (22%; P<0.05).

Interestingly, previous experiments found a significant (34%) increasein K_(s) in normal, innervated gastrocnemius muscles from mature ratsinjected with clenbuterol. No such change in K_(s) was observed in thepresent study. The previous experiments differed from the present studyin several respects such as animal age, mode of administration and doseof clenbuterol, and in particular the time-interval after drugadministration at which K_(s) was measured. It has been suggested thatit is the latter difference, measurement of K_(s) coinciding with thepeak of the thermogenic response within 1 hr of the daily injection,which accounts for the difference in data.

It might be argued that the difference in response of innervated anddenervated muscle to clenbuterol was associated with increased numbersof beta-adrenergic receptors in the denervated muscle membrane. Thisdoes not, however, appear to be the case. Even in the presence of themixed beta-antagonist propranolol (2 mg/d equivalent to 100×excess ofclenbuterol, a dose which has been shown to block the cardiachypertrophy and the reduction in body fat), clenbuterol remainseffective in reversing the muscle protein loss following denervation.

A factor from sciatic nerve has been identified, which, when injectedintramuscularly, will stimulate the growth of denervated muscles.Although it is tempting to speculate that the effects of clenbuterol ondenervated muscle might in some way be mediated by this myotrophicfactor this is clearly an inadequate explanation for the proteinanabolic actions of the drug. For example, the previously isolatedmyotrophic factor was without effect on innervated muscle whereasclenbuterol, in functionally innervated muscle, is markedly anabolicthrough alterations in protein breakdown. The changes in translationalefficiency and capacity in clenbuterol treated denervated muscles arehowever similar to the responses of other cell types to specific peptidegrowth factors. Restoration of translational efficiency has beenobserved as a response to many stimuli, including refeeding afterfasting, and there may be several or indeed many signals which can berecognised. In normal muscles one such signal may be produced from thefunctional nerve which fully activates the system. Further stimuliinduced either directly or indirectly by clenbuterol may selectivelyalter proteolysis.

In conclusion we have demonstrated that the novel response of denervatedmuscle to the beta-agonist clenbuterol involves at least two mechanisms.The first involves, in the down-regulated denervated state, changes intranslational efficiency and capacity which are reminiscent of theclassical pleiotypic responses of cells to growth factors. The second isa specific reduction in protein breakdown. In the innervated state thefirst effect is not observed because the muscles have already respondedto other stimuli, whereas the second is manifested. Many otherphysiological stimuli enhance protein gain through mechanisms whichstimulate primarily protein synthesis but also degradation. However, thespecial characteristics of clenbuterol, and its analogues, appear tooffer exciting advances both in manipulating muscle composition andgrowth as well as being potentially valuable tools in the study andtreatment of myogenic disorders.

                  TABLE 1                                                         ______________________________________                                        (Section 6):                                                                  Compositional effect of clenbuterol on                                        innervated and denervated rat soleus muscle                                   Day after                                                                              Innervated limb                                                                              Denervated limb                                       Clenbuterol                                                                            Control  + Clen.   Control                                                                              + Clen.                                    ______________________________________                                               Protein content (mg)                                                   0        5.3                3.0***                                            0.5      5.7      5.6       3.2*** 3.5*                                       1.0      6.1      6.3       3.4*** 3.5**                                      1.5      6.1      6.1       2.9*** 3.6*, +                                    3.0      6.7      7.3+      3.0*** 3.9**, ++                                  7.0      9.7      12.1+++   3.5*** 6.0***, +++                                S.E.D. for effect of clenbuterol 0.3082                                       S.E.D. for effect of nerve status 0.2425                                             RNA content (μg)                                                    0        94                 52***                                             0.5      92       93        61***  66***                                      1.0      104      104       61***  72***, +                                   1.5      100      106       52***  86***, +++                                 3.0      108      121+      56***  91***, +++                                 7.0      132      162+++    60***  98***, +++                                 S.E.D. for effect of clenbuterol 5.574                                        S.E.D. for effect of nerve status 5.170                                              RNA Protein (μg/mg)                                                 0        17.5               17.4                                              0.5      16.1     16.5      19.0*  18.7                                       1.0      17.1     16.5      18.4   22.5***, ++                                1.5      16.2     17.3      18.0   23.6***, +++                               3.0      16.1     16.5      18.99* 23.4***, ++                                7.0      13.6     13.4      17.1** 16.4*                                      S.E.D. for effect of clenbuterol 1.389                                        S.E.D. for effect of nerve status 1.222                                       For effect of clenbuterol +++p < 0.005, ++p < 0.01,                           +p < 0.05                                                                     For effect of nerve status ***p < 0.005, **p < 0.01, *p < 0.05                (Protein and RNA contents and the ratio RNA:protein in the                    innervated and denervated soleus muscles of rats subjected to                 unilateral sciatic denervation)                                               ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        (Section 6)                                                                   Metabolic effects of clenbuterol on innervated and denervated rat             soleus muscle                                                                 ______________________________________                                                Innervated limb                                                                           Denervated limb                                           Day after Control  + Clen-  Control                                                                              + Clen-                                    Clenbuterol                                                                             diet     buterol  diet   buterol                                    ______________________________________                                                K, (%/day)                                                            0         27.7              19.8***                                           0.5       29.3     26.5     21.9** 25.9                                       1.0       24.6     23.8     20.3   32.4***, +++                               1.5       25.1     27.1     23.5   36.6***, +++                               3.0       27.1     27.1     25.8   31.6+                                      7.0       18.1     19.6     18.6   21.8                                       S.E.D. for effect of clenbuterol 2.691                                        S.E.D. for effect of nerve status 2.279                                               K.sub.3 /RNA (g prot. synthesised/g RNA)                              0         16.5              11.2***                                           0.5       18.1     16.0     11.9***                                                                              14.1                                       1.0       14.4     14.3     11.1*  14.5++                                     1.5       15.7     15.9     13.2   15.4                                       3.0       16.9     16.6     14.8   13.6*                                      7.0       13.4     14.6     10.9   13.3*                                      S.E.D. for effect of clenbuterol 1.732                                        S.E.D. for effect of nerve status 1.262                                       K.sub.g and calculated K.sub.d                                                Innervated limb     Denervated limb                                           K.sub.g  K.sub.d K.sub.g                                                                              K.sub.d                                                                              K.sub.g                                                                          K.sub.d                                                                             K.sub.g                                                                            K.sub.d                          Control      + Clen-    Control   + Clen-                                     diet         buterol    diet      buterol                                     ______________________________________                                        0-1 d 14.0   13.2    17.2 8.6   0   20.7  15.4 10.6                           1-3 d 9.5    16.1    14.7 11.3  0   23.2  11.4 22.1                           3-7 d 11.0   11.6    12.4 11.0  0   22.2  10.9 15.8                           ______________________________________                                         For the effect of clenbuterol +++p < 0.005 ++p < 0.01 +p < 0.05               For the effect of nerve status ***p < 0.005 **p < 0.01 *p < 0.05              (The fractional rate of protein synthesis (K.sub.3) in both the innervate     and denervated soleus muscles of the same rats as described in Table 1.       The fractional rate of protein deposition (K.sub.g) was based on the          change in muscle protein between the periods specified. The fractional        rate of protein degradation (K.sub.d) was estimated as the difference         between the mean values of K.sub.g and K.sub.3. It was assumed that the       small amount of growth in the denervated limbs of control fed rats was no     significantly different from zero. The resultant value is taken to be an      indication of the rate of protein degradation in these muscles over the       time period specified.)                                                  

Section 7 The Effect of Clenbuterol on Clinical Course and NitrogenBalance in Patients with Cancer Cachexia BACKGROUND

Clenbuterol is a beta(2) sympathomimetic drug which has been used in thetreatment of bronchospasm in animals and man. Clinical trials have showna similar efficacy and side-effect profile to salbutamol or terbutalinewhich are the most popular beta(2) agonists in use in the United Kingdom(where clenbuterol is not currently available for clinical use).

Recent work has shown that clenbuterol has three additional effects onmuscle protein which are not seen with salbutamol or terbutaline.Firstly clenbuterol can specifically increase the rate of skeletal andcardiac muscle growth in young animals; there is a slight reduction ingrowth rate of the viscera of drug-treated animals with respect tocontrol animals. Secondly clenbuterol limits the atrophy of skeletalmuscle in denervated limbs in rats. Thirdly in animals treated withclenbuterol after the development of denervation atrophy, reversal ofthe atrophic changes occur with a simultaneous increase in contractilestrength.

The need for a drug which can selectively promote growth or limitotherwise irresistible wasting exists in many pathological situationsaffecting enormous numbers of patients and it is therefore importantthat potential clinical applications of the protein anabolic effects ofclenbuterol are made available at the earliest proof of efficacy. Ofparticular importance in relation to maintenance of muscle mass is theobservation that loss of lean body tissue to approximately 80-85% of theideal figure almost always results in a fatal outcome. This situation isfrequently encountered in patients with neoplastic disease andassociated cachexia with its attendant muscle weakness. Treatment atthis stage has little to offer other than analgesia but patients in thisposition could benefit in terms of subjective well-being and longersurvival if lean tissue wasting could be slowed or arrested by a drugsuch as clenbuterol. In view of the slight limitation on visceral growththere is no indication that tumour growth would be enhanced by the drugand indeed there is a possibility that it may be reduced due to thepreferential anabolic effect on skeletal muscle.

OUTLINE OF PROPOSED PROOF OF EFFICACY OF CLENBUTEROL:

A preliminary study of the effect of clenbuterol in cancer cachexia ishereby proposed. In the first instance such a study would have to besimple in design and objectives as it would be unethical to subjectterminally ill patients to arduous investigation without more positiveevidence of likely therapeutic benefit. Nevertheless it is importantthat the study is carried out and it should not prove difficult for thesuppliers of clenbuterol to obtain the necessary Clinical TrialExemption Certificate from the Committee for the Safety of Medicines toallow the use of clenbuterol in a group of patients who have little tolose and possibly much to gain. This certification would then allow anapproach to the relevant Ethical Committee who are likely to approve theproposed protocol. Due to the varied clinical forms, manifestations anddisease course in patients with established malignancy it would benecessary to study reasonable numbers of patients (say 30-40) in orderto allow a useful assessment of use of clenbuterol in practice. Patientswith solid tumours, a documented weight loss of more than 10% and whoare receiving only palliative therapy would be studied. The study designwould be dual with an initial parallel, placebo controlled phasefollowed, for those who are willing and able to continue by a secondcrossover phase. Those entering the study would have a 3-day run-inperiod before randomisation to receive either clenbuterol 20microgrammes three times daily or placebo for 7 days followed by a 7-daywashout period; those able to continue would then take placebo orclenbuterol for a further 7 days finishing with a 3-day run-out period.Protein wasting would be assessed simply by measurement of daily urinarynitrogen losses throughout the study. Clinical assessment, diagnosticclassification, previous and current therapy would be noted. Dietarydiaries would be kept to monitor any changes in food intake during thestudy, subjective assessment of well-being and debility made usingvisual analogue scales and any adverse effects of trial medicationrecorded. In addition, when appropriate to each patient's condition,simple tests of active muscle function could be made at intervalsthroughout the study. Basic anthropometric measurements (height, weightand skinfold thickness) would also be recorded at intervals.

The current incidence of cancer cachexia in the available populationwould enhance completion of the study within approximately 12 months.

Section 8 The Effect of Clenbuterol on Skeletal Muscle Development inRowett Hodded Lister Rats BACKGROUND

Previous studies in rats have demonstrated that the effects ofclenbuterol are influenced or determined by the physiological state ofthe muscle or its fibre type. In several respects (eg membraneproperties, isozyme patterns, protein turnover rates) denervated musclehas similarities with that of the foetus or neonate. Our studies haveshown that clenbuterol was particularly effective as an anabolic agentin denervated muscle. The response was of greater magnitude and morerapid than in innervated, growing, fully differentiated muscle.Furthermore, the mechanism by which this response was elicited was alsodifferent from that in innervated muscle. Whereas clenbuterol gave riseto little or no change in the fractional rate of protein synthesis innormal innervated muscle, its effect on denervated muscle was tonormalise translationed efficiency (g protein synthesised/g RNA) andsignificantly increase the fractional rate of protein synthesis. Thesedata suggest that the undifferentiated or immature muscle may beparticularly responsive to exposure to beta-two agonists, or morespecifically clenbuterol and its analogues. It is well documented thatthe foetus is sensitive to specific signals at particular stages ofdevelopment, eg single dose or short term administration ofgonadotrophins to neonatal rats can produce marked changes in bodycomposition which persist through to adulthood. If clenbuterol exertsits muscle-specific effects by altering the fundamental properties ofthe muscle (for example at the plasma membrane) then modificationsinduced early in development may be persistent. An investigation of sucha possibility requires that the drug (or active factor associated withthe drug administration) crosses the placenta for foetal impringing oris secreted in the milk for dosing the neonate.

Thus the aim of this work was to examine the effect of clenbuterol onskeletal muscle development and to assess the possibility thatinterference with the early pattern of development may result in apermanent alteration of the growth pattern.

Two areas of focus were chosen:

a. Ante-natal muscle development

b. Muscle development from birth to post-weaning

c. Antenatal Development in Soleus and EDL Muscles of Rats:

It is well established that the temporal sequences of events duringmyogenesis is of crucial importance in establishing the geneticexpression of the individual muscle cells, and hence the muscle as awhole. Myotubes start to form in the foetus at around 15 d gestation, atime at which there are few neural contacts. The first experiment wastherefore designed to expose the foetus to clenbuterol from 12 dgestation and to sample the muscles at 19 d gestation, at which timeneuromusclar junctions can be found and the muscle is beginning tomature.

Pregnant dams (mother animals) were fed control diet plus clenbuterol (2mg/kg) from 12 d gestation. Whole single soleus and EDL muscles wereisolated from the 19 d foetuses and examined at both light and electronmicroscopic levels. Muscles from clenbuterol-treated foetuses possessedprimitive differentiated motor endplates on both primary and secondarygeneration myotubes. In control muscles, however, it was not common tofind endplates on secondary generation myotubes. Moreover, it wasevident that in the clenbuterol-treated myotubes fusion had occurred toa greater extent than in the controls. The myofibrillar proteins showeda greater degree of spatial organisation, and the tubular systemsappeared to be more extensively organised than in controls.

Overall, the microscopic results indicated that clenbuterol treatment offoetal rat muscle in utero had resulted in the advancement of musclefibre fusion. This contention was supported by the biochemical analyses.Since single muscles were too small for accurate assessments,determinations were made on the whole posterior distal muscle group.

The major results were:

i. The drug increased the weight (P<0.05) and protein content (P<0.01)of the foetal hearts.

ii. Skeletal muscle wet weights, and protein content and concentrationswere significantly (P<0.005) reduced in clenbuterol-treated foetusescompared to the controls.

iii. DNA concentration was significantly (P<0.005) elevated inclenbuterol-treated groups, but RNA concentration was not significantlydifferent from control values.

iv. Both total RNA and DNA contents were significantly (P<0.005)reduced.

These results, while supporting the concept that clenbuterol treatmentadvances fusion of myoblasts, need to be interpreted with caution.

Unexpectedly a considerable anabolic effect of clenbuterol was observedin the pregnant dams. Thus it is possible that some penalty was directedat the foetuses; and in particular at the foetal skeletal muscle sincethe agonist effect on heart weight is expressed as in weaning animals.

In the dams it was found that:

i. there was a 15-30% increase in weight of individual muscles (as afraction of body weight) in the clenbuterol-treated dams with respect tothe controls.

ii. the heart weights were increased in the treated animals by 10-20%.

However, there was no significant differences with respect to eithermean foetal number or mean foetal weight between control and clenbuterolfed groups.

These results then suggested that clenbuterol was accelerating myotubeformation at the expense of cell division. It was therefore important toestablish the effect of clenbuterol on muscle fibre number. Consequentlya further set of experiments were carried out to examine the effect ofcontinuous exposure to the drug from 12 d gestation through parturitionto weaning. We observed that while the presence of clenbuterol in thediet did not inhibit the final outcome or success of parturition, theonset of labour was delayed by approximately 12 hours in comparison withcontrol dams.

As with dams killed prior to parturition, dams fed clenbuterol untilweaning showed an increase in heart weight of approximately 7% and inmuscle weight of 20-30%.

As before, pregnant dams were fed diet containing clenbuterol (2 mg/kg).After parturition litters were adjusted to 8 pups (4 of each sex) andthe pups maintained with their mothers until weaning. The results fromthe weanling rats exposed to clenbuterol continuously from 12 dgestation were particularly interesting. As observed in the study offoetal muscle, treatment with clenbuterol resulted in a significantreduction in muscle weight. Histological examination, however, revealedthat there was a significant (P<0.05) reduction in total fibre number inboth soleus and EDL muscles from treated animals. Furthermore thecross-sectional area of these fibres was considerably greater than thosefrom control animals. These results were in contracts to those fromweanling rats exposed to clenbuterol for 4 days only: the fibrehypertrophy was not accompanied by a reduction in fibre number.

Thus, this data together with that from the foetuses indicated that:

i. clenbuterol was accelerating myotube/myofibre formation at theexpense of cell division;

ii. as a result of which, there appeared to be a permanent change infibre number and size;

iii. in other words that clenbuterol has two effects:

1. in foetal muscle it alters fibre number, presumably under genotypiccontrol; and

2. in adult muscle it alters fibre size, presumably under phenotypiccontrol.

b. Effects of Neonatal Exposure

The observations made with animals exposed first to Clenbuterol in uteroshowed the common pattern of increased fusion and reduced fibre number.In consequence these two effects approximately cancelled out, so thepups did not show a muscle weight or protein anabolic response asobserved for post-weaning animals.

One hypothesis that follows from this is that Clenbuterol acts through asingle system, which has a developmental aspect, and that muscle gain isrelated to maturity; either of the muscle or associated receptorpopulations or of the nerve supply. A second hypothesis is that thefoetal/neonatal and the post-weaning responses do, in fact, reflectClenbuterol interaction with two, at least, different mechanisms.

It was therefore appropriate to examine the stage at which the ratbecomes sensitive to Clenbuterol, when assessed in terms of muscleprotein anabolism. In the first study attempted, pups were born to damsmaintained on control diet. Litters were adjusted to 8 pups (4 of eachsex), and half the mothers transferred to control diet plus Clenbuterol(2 mg/kg). At 18 days the pups were analysed. Pups from dams givenClenbuterol were 9% lighter than controls while absolute muscle weightswere decreased by 11-13%. On a body weight basis heart mass wasincreased by 7% while liver size decreased by 7%. In plantaris musclestotal protein from Clenbuterol-treated animals were lower by 7%, RNA wasunaltered while DNA was decreased by 10%. Essentially similar data wereobtained for soleus muscle.

This experimental design assumed that, first, Clenbuterol would be madeavailable to the pups from the milk of the dam in sufficient quantitiesand, second, that the drug would not alter the lactational output. Inpractice the slight effect on relative heart mass did indicate that someClenbuterol, at lest, was made available through the mammary gland.However, from the daily weights of pups it was clear that there was amajor check in growth during the first 48 h that the dams received thedrug--this was probably due to reduced milk secretion (the intake ofdams declined during this short period). Furthermore, analysis of thedams for body composition at the end of the study showed, as for thepregnant state, that muscle growth was promoted by a 4-12% inClenbuterol-treated animals; heart mass was unaltered. Clenbuterol thusappeared to have over-ridden, at least partially, the supply ofnutrients to the mammary gland. As blood flow, and thus probably heartsize, is already elevated during lactation a potential re-distributionto other tissues might be achieved without a net increase in overallcardiac output and without the requirement for elevated heart mass.

These findings indicated a different experimental approach was required.In consequence, rather than including the drug within the diet of themother, pups were dosed orally, twice a day, with appropriate amounts ofClenbuterol (fixed as 200 microgrammes/kg body weight; this is the sameas used in the growth studies described in a previous section above).The design of the experiment was otherwise similar to the aboveantenatal experiment, ie litters culled to 8 (4 of each sex) and then atstaggered intervals from 4 days onwards half the litter (2 of each sex)received Clenbuterol while the other half received vehicle (water). Thisdesign was to reduce scatter due to inter-litter variation. Two litterswere applied to each treatment, in which Clenbuterol was administeredfrom either day 4, day 8, day 12, day 16 or day 19 (weaning) and allpups were analysed at day 25.

No differences in body weight were found. All Clenbuterol-treatedanimals showed increases in heart weight (9-15%). Only in animals giventhe drug from day 19 onwards were differences in muscle weight (+10%)observed. Previous treatment, ie dosage at times between days 4-25 anddays 16-25 had no effect on muscle weight. Muscle protein content,however, was increased equally with all treatments from day 8 onwards(magnitude of increase 11-13%). Total RNA was unaffected by Clenbuterolbut, in common with the foetal studies, there were again significantreductions (P<0.01) in DNA content. For animals receiving the drug at 16days or earlier the decrease was 9-13%, whereas with treatment at days19-25 the decline was only 5%.

The effect on protein gain, therefore, was occurring most activelyduring the immediate post-weaning period (days 19-25) and that earlierexposure did not make a significant contribution. Two explanations couldaccount for these observations.

1. The earlier exposure had occurred at a time in which the mechanismleading the muscle protein gain was inactive. Alternatively the systemmay have been present, but inactive, and was desensitised ordown-regulated by early exposure to the drug.

2. The promotion of early fibre fusion, noted for embryonic muscle,continued in early to late suckling with consequent larger fibres butfewer in number so that the potential for extra muscle growth waslimited.

This experiment has been repeated, in order to focus on the temporalsequence of events involved. The design was in principle thatillustrated above, but now the time intervals of exposure were alteredto 2 day increments, applied from days 14-24 with all animals analysedat day 28. Again maximum responses were observed for animals treatedfrom either day 20 or day 22, it was reduced with treatment from day24-28. Responses for animals dosed from day 14 to day 18 were 50-100%lower than the optimal growth of muscle protein mass. Heart weightincreases (16-24%) were similar for all groups.

This experiment again confirms that the weaning period represents a timeof great sensitivity for the anabolic action of the drug. At this stageof development considerable changes occur in gut morphology andmetabolism. In addition, muscle tissue also becomes especially sensitiveto specific hormones involved in the regulation of protein metabolism,for example, the glucocorticoids. The potential interaction ofClenbuterol with either systemic or gut hormones needs to beinvestigated further.

While the above experimental information refers particularly to rats, itis considered possible that the same or similar effects would beobserved in other mammalian animals and in humans, and possibly to acertain extent in non-mammalian animals.

I claim:
 1. A method of alleviating or reversing loss of function ofstriated muscle arising from at least one illness in the group ofillnesses consisting of:a) muscle disease; b) peripheral nervous systemdisease; and c) central nervous system disease, wherein said methodcomprises the step of administering an effective amount of abeta-adrenergic agonist and a suitable carrier in diluent, wherein saidbeta-adrenergic agonist is selected from the group consisting ofclenbuterol, an acid addition salt of clenbuterol, and an analogthereof.
 2. A method as claimed in claim 1 wherein the muscle disease ismuscular dystrophy.